it is a heterotetramer of the two subunits of kinesin light chain 1 ( klc1 ) and two subunits of kinesin heavy chains ( khcs ) .
kif5b , a khc , contains a motor domain at its n - terminal , which hydrolyzes atp and converts the chemical energy to mechanical energy for movement along microtubules .
its c - terminal region contains the dimerization domain that interacts with klc1 , which associates with cargos .
recently , kif5b was shown to transport the nuclear transcription factor c - myc for proteosomal degradation in the cytoplasm .
three methodologies ( chemical inhibitor , sirna/ shrna and dominant negative mutant ) were used to inhibit kinesin-1 function .
for the last methodology , c - myc was only affected by the dominant negative mutant of kif5b , but not by that of another related kif5a motor protein , suggesting that the mutant does not exert general effects on the intracellular components ( like microtubule disruption ) or on protein aggregation .
the dominant negative mutant of kif5b also did not affect another transcription factor , suggesting that it does not exert general effects on transcription factors .
rather , it suggests that the dominant negative mutant exerts specific effects on its cargos .
they were mainly used to demonstrate the effects of the mutants on the subcellular localizations of their cargos or on cellular functions .
less emphasis was put on the spatial relationship between the mutants and the cargos affected by them . however , in some incidences , the mutants were observed to co - localize with their cargos . the interaction between kif5b and
its associated proteins was previously confirmed by the in vivo yeast two - hybrid assay and biochemical pull - down assays such as co - immunoprecipitation and in vitro pull - down assays . in this article ,
the method makes use of a motorless kif5b mutant that acts as a dominant negative mutant .
the tagging of wild - type and motorless kif5b mutant with the fluorescent protein tdtomato enables their visualization by fluorescence microscopy .
the tagged kif5b proteins can be co - expressed with a candidate protein fused to a different fluorescent protein with spectral properties suitably separated from the kif5b tag .
induction of aggregation of the candidate protein by the motorless kif5b mutant will confirm that the candidate protein is an in vivo cargo of kif5b .
furthermore , the tdtomato - tagged kif5b proteins can be expressed alone in the cells to study their effects on the endogenous cargo proteins .
later , immunofluorescence microscopy is conducted in which the transfected cells are fixed and stained with a specific antibody against the endogenous candidate protein , followed by an appropriate secondary antibody conjugated with a fluorescent dye . in this case , the endogenous candidate protein at its physiological level is studied .
amplify the cdnas for the human wild - type and motorless kif5b proteins using the primers in table 1 , taq dna polymerase ( 5 units for 100 l ) , dntp mix ( 2 mm for each deoxynucleotide ) and its 10x buffer for 30 cycles .
each cycle consists of a denaturation step ( 95 c for 30 sec ) , an annealing step ( 45 c for 30 sec ) and an extension step ( 72 c for 3 min ) .
transfer the aqueous solution to a new tube and extract with an equal volume of chloroform.spin at 18,000 x g in a microcentrifuge at room temperature for 0.5 min .
transfer the aqueous solution to a new tube.mix the aqueous solution with one tenth volume of 3 m na - acetate and two volumes of absolute ethanol.spin at 18,000 x g in a microcentrifuge at room temperature for 5 min . discard the aqueous solution.wash the dna pellet with two volume of 75% ethanol .
discard the aqueous solution and air dry the dna pellet at room temperature for 5 min .
transfer the aqueous solution to a new tube and extract with an equal volume of chloroform .
mix the aqueous solution with one tenth volume of 3 m na - acetate and two volumes of absolute ethanol .
wash the dna pellet with two volume of 75% ethanol . discard the aqueous solution and air dry the dna pellet at room temperature for 5 min .
digest the amplified products in a final volume of 40 l with restriction enzymes sali ( 10 units ) and bamhi ( 10 units ) and 4 l of their 10x buffer at 37 c for two hr . resolve the digested products in an agarose gel ( 1.0% at 100 v ) containing ethidium bromide ( 0.2 g / ml ) .
weigh the agarose gel containing the dna fragment . then dissolve it in the solubilization buffer ( 300 l for 0.1 g ) at 37 c for about 20 min .
add the resulted solution to a column and spin in a microcentrifuge at room temperature for 5 sec .
elute the dna fragment with 50 l water by repeating step 1.5 . estimate the concentration of the dna fragment by running an aliquot of it against a dna size ladder in an agarose gel ( 1.0% at 100 v ) containing ethidium bromide ( 0.2 g / ml ) .
ligate the purified products ( about 100 ng ) with the ptdtomato - c1 vector ( about 100 ng ) previously digested with the same restriction enzymes in 10 l using t4 dna ligase ( 2,000 units ) and 1 l 10x ligation buffer at room temperature for 16 hr .
note : the wild - type and motorless mutant contains amino acids from 2 to 963 and 584 to 963 , respectively .
a smaller motorless kif5b mutant was used for the present studies to increase its expression levels . for live - cell or indirect fluorescence imaging with magnification at or below 40x , seed 0.2 - 0.3 million hela cells in 1 ml complete medium [ dulbecco 's modified eagle medium ( dmem ) with 10% fetal bovine serum ( fbs ) ] in each well of a six - well plate .
grow at 37 c with 5 % co2 . for indirect immunofluorescence studies with magnifications above 40x , wash cover glasses ( 18 mm x 18 mm ; 1.5 thickness ) in absolute ethanol briefly and air - dry them in the wells of a six - well plate , inside a biosafety cabinet to avoid contamination .
seed 0.2 - 0.3 million cells in 1 ml complete dmem medium in each well of a six - well plate . 
 
seed 0.2 - 0.3 million cells in 1 ml complete dmem medium in each well of a six - well plate .
preparation of transfection complex the next day , inside a biosafety cabinet , dilute 0.6 g ( total ) of the expression plasmid for tdtomato - tagged wild - type or motorless kif5b in the presence or absence of an expression plasmid for a cargo candidate protein ( ptagcfp - c - myc ) with 0.1 ml transfection medium in a 1.6 ml microcentrifuge tube .
note : the vector ptagcfp - c - myc expresses the tagcfp - tagged c-myc.add 1.8 l of the transfection reagent to 0.1 ml transfection medium in another 1.6 ml microcentrifuge tube .
typically , prepare a master mix of diluted transfection reagent enough for 12 samples.incubate for 5 min , at room temperature.add the diluted 0.1 ml transfection reagent to the diluted 0.1 ml dnas .
spin the tubes for 5 sec in a microcentrifuge.incubate for 45 min , at room temperature . 
  the next day , inside a biosafety cabinet , dilute 0.6 g ( total ) of the expression plasmid for tdtomato - tagged wild - type or motorless kif5b in the presence or absence of an expression plasmid for a cargo candidate protein ( ptagcfp - c - myc ) with 0.1 ml transfection medium in a 1.6 ml microcentrifuge tube .
note : the vector ptagcfp - c - myc expresses the tagcfp - tagged c - myc .
add 1.8 l of the transfection reagent to 0.1 ml transfection medium in another 1.6 ml microcentrifuge tube . typically , prepare a master mix of diluted transfection reagent enough for 12 samples .
washing of cells wash the seeded cells three times with phosphate buffered saline ( pbs ) during the incubation period for the formation of the transfection complex.replace the medium of the seeded cells in each well with 0.8 ml pre - warmed transfection medium .
wash the seeded cells three times with phosphate buffered saline ( pbs ) during the incubation period for the formation of the transfection complex .
replace the medium of the seeded cells in each well with 0.8 ml pre - warmed transfection medium .
transfection of cells after incubation of 45 min , add 0.2 ml of the dna / transfection reagent complex ( prepared in step 2.3 ) dropwise to each well of the plate.rock the 6 well plate gently for 5 sec , before they are returned to the incubator at 37 c.after 6 - 8 hr , replace the medium with the complete dmem medium . 
 
after incubation of 45 min , add 0.2 ml of the dna / transfection reagent complex ( prepared in step 2.3 ) dropwise to each well of the plate .
rock the 6 well plate gently for 5 sec , before they are returned to the incubator at 37 c .
after 6 - 8 hr , replace the medium with the complete dmem medium . for live - cell fluorescence imaging 
 after an additional 16 hr of incubation at 37 c , add the dna intercalating stain hoechst 33342 to the medium to a final concentration of 1 m . note : hoechst 33342 is a cell - permeable , dna intercalating dye that stains the cell nuclei.incubate the transfected cells for 10 min in the incubator at 37 c before examining them for the expression of fluorescent proteins by fluorescent microscopy using a 40x objective .
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively . note : if the background is high due to auto - fluorescence of the complete dmem medium , medium without phenol red is used . 
 
after an additional 16 hr of incubation at 37 c , add the dna intercalating stain hoechst 33342 to the medium to a final concentration of 1 m . note : hoechst 33342 is a cell - permeable , dna intercalating dye that stains the cell nuclei .
incubate the transfected cells for 10 min in the incubator at 37 c before examining them for the expression of fluorescent proteins by fluorescent microscopy using a 40x objective .
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively . note : if the background is high due to auto - fluorescence of the complete dmem medium , medium without phenol red is used .
preparation of paraformaldehyde solution weigh paraformaldehyde powder ( 4 g per 100 ml pbs ) and add it to pbs . note : paraformaldehyde is a flammable solid and a potential cancer hazard .
therefore , it is important to avoid contact with or inhalation of paraformaldehyde.add naoh to the solution ( 150 l 10 n naoh/100 ml).keep the solution at 37 to 42 c for 2 - 3 hr with occasional shaking.adjust ph to 7.0 by adding glacial acetic acid to the solution ( about 75 l/ 100 ml ) after the paraformaldehyde powder is dissolved . 
 
weigh paraformaldehyde powder ( 4 g per 100 ml pbs ) and add it to pbs .
keep the solution at 37 to 42 c for 2 - 3 hr with occasional shaking .
adjust ph to 7.0 by adding glacial acetic acid to the solution ( about 75 l/ 100 ml ) after the paraformaldehyde powder is dissolved .
target proteins staining after further incubation for 16 hr at 37 c , wash the transfected cells at room temperature once with pbs by swirling the six - well plate for 5 sec.fix the cells with 1 ml of freshly prepared , 4% paraformaldehyde solution per well .
incubate at room temperature for 30 min.wash the cells with pbs once by swirling for 5 sec the six - well plate .
discard the solution.incubate the cells with 1 ml of 0.1% triton - x 100 ( in pbs ) at room temperature for 30 min .
the detergent triton - x 100 will permeabilize the cell membrane to allow access of antibodies to their intracellular targets.wash the cells with pbs four times by swirling the six - well plate for 5 sec each time .
discard the solution after each wash.incubate the cells with 1 ml of primary antibodies ( c - myc rabbit antibody or p53 mouse antibody ; 0.1 g / ml ) in a 10% fbs in pbs solution at room temperature by rocking for 4 hr.wash with pbs four times by swirling the six - well plate for 5 sec each time .
discard the solution after each wash.incubate with 1 ml of fluorescent dye - conjugated secondary antibodies ( alexa fluor 488-conjugated anti - rabbit or anti - mouse igg antibody ; 0.5 g / ml ) in 10% fbs in pbs at room temperature in the dark by rocking for 2 hr.wash with pbs four times by swirling the six - well plate for 5 sec each time .
discard the solution after each wash . 
  after further incubation for 16 hr at 37 c , wash the transfected cells at room temperature once with pbs by swirling the six - well plate for 5 sec .
fix the cells with 1 ml of freshly prepared , 4% paraformaldehyde solution per well .
wash the cells with pbs once by swirling for 5 sec the six - well plate .
incubate the cells with 1 ml of 0.1% triton - x 100 ( in pbs ) at room temperature for 30 min .
the detergent triton - x 100 will permeabilize the cell membrane to allow access of antibodies to their intracellular targets .
wash the cells with pbs four times by swirling the six - well plate for 5 sec each time .
incubate the cells with 1 ml of primary antibodies ( c - myc rabbit antibody or p53 mouse antibody ; 0.1 g / ml ) in a 10% fbs in pbs solution at room temperature by rocking for 4 hr .
wash with pbs four times by swirling the six - well plate for 5 sec each time .
incubate with 1 ml of fluorescent dye - conjugated secondary antibodies ( alexa fluor 488-conjugated anti - rabbit or anti - mouse igg antibody ; 0.5 g / ml ) in 10% fbs in pbs at room temperature in the dark by rocking for 2 hr . wash with pbs four times by swirling the six - well plate for 5 sec each time . discard the solution after each wash .
nuclear staining and mounting incubate the cells with the 1 ml of dna intercalating dye 4',6-diamidino-2-phenylindole ( dapi ; 0.5 g / ml ) in pbs at room temperature , in dark for 10 min .
note : dapi is a dna intercalating dye that stains the cell nuclei.apply 10 l of mounting solution onto each microscope slide .
mount each cover glass over the mounting solution on a microscope slide.incubate at room temperature in the dark overnight.seal the edges of the cover glasses with nail polish .
place in the dark inside a fume hood overnight to remove the nail polish fumes . 
 
incubate the cells with the 1 ml of dna intercalating dye 4',6-diamidino-2-phenylindole ( dapi ; 0.5 g / ml ) in pbs at room temperature , in dark for 10 min .
place in the dark inside a fume hood overnight to remove the nail polish fumes .
fluorescence microscopy 
 next day , examine the cells by fluorescent microscopy using a 40x objective .
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively . 
  next day ,
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively .
amplify the cdnas for the human wild - type and motorless kif5b proteins using the primers in table 1 , taq dna polymerase ( 5 units for 100 l ) , dntp mix ( 2 mm for each deoxynucleotide ) and its 10x buffer for 30 cycles .
each cycle consists of a denaturation step ( 95 c for 30 sec ) , an annealing step ( 45 c for 30 sec ) and an extension step ( 72 c for 3 min ) .
transfer the aqueous solution to a new tube and extract with an equal volume of chloroform.spin at 18,000 x g in a microcentrifuge at room temperature for 0.5 min .
transfer the aqueous solution to a new tube.mix the aqueous solution with one tenth volume of 3 m na - acetate and two volumes of absolute ethanol.spin at 18,000 x g in a microcentrifuge at room temperature for 5 min . discard the aqueous solution.wash the dna pellet with two volume of 75% ethanol .
discard the aqueous solution and air dry the dna pellet at room temperature for 5 min .
transfer the aqueous solution to a new tube and extract with an equal volume of chloroform .
mix the aqueous solution with one tenth volume of 3 m na - acetate and two volumes of absolute ethanol .
wash the dna pellet with two volume of 75% ethanol . discard the aqueous solution and air dry the dna pellet at room temperature for 5 min .
digest the amplified products in a final volume of 40 l with restriction enzymes sali ( 10 units ) and bamhi ( 10 units ) and 4 l of their 10x buffer at 37 c for two hr . resolve the digested products in an agarose gel ( 1.0% at 100 v ) containing ethidium bromide ( 0.2 g / ml ) .
weigh the agarose gel containing the dna fragment . then dissolve it in the solubilization buffer ( 300 l for 0.1 g ) at 37 c for about 20 min .
add the resulted solution to a column and spin in a microcentrifuge at room temperature for 5 sec .
elute the dna fragment with 50 l water by repeating step 1.5 . estimate the concentration of the dna fragment by running an aliquot of it against a dna size ladder in an agarose gel ( 1.0% at 100 v ) containing ethidium bromide ( 0.2 g / ml ) .
ligate the purified products ( about 100 ng ) with the ptdtomato - c1 vector ( about 100 ng ) previously digested with the same restriction enzymes in 10 l using t4 dna ligase ( 2,000 units ) and 1 l 10x ligation buffer at room temperature for 16 hr .
note : the wild - type and motorless mutant contains amino acids from 2 to 963 and 584 to 963 , respectively .
a smaller motorless kif5b mutant was used for the present studies to increase its expression levels .
for live - cell or indirect fluorescence imaging with magnification at or below 40x , seed 0.2 - 0.3 million hela cells in 1 ml complete medium [ dulbecco 's modified eagle medium ( dmem ) with 10% fetal bovine serum ( fbs ) ] in each well of a six - well plate
. grow at 37 c with 5 % co2 . for indirect immunofluorescence studies with magnifications above 40x , wash cover glasses ( 18 mm x 18 mm ; 1.5 thickness ) in absolute ethanol briefly and air - dry them in the wells of a six - well plate , inside a biosafety cabinet to avoid contamination .
seed 0.2 - 0.3 million cells in 1 ml complete dmem medium in each well of a six - well plate . 
 
seed 0.2 - 0.3 million cells in 1 ml complete dmem medium in each well of a six - well plate .
preparation of transfection complex the next day , inside a biosafety cabinet , dilute 0.6 g ( total ) of the expression plasmid for tdtomato - tagged wild - type or motorless kif5b in the presence or absence of an expression plasmid for a cargo candidate protein ( ptagcfp - c - myc ) with 0.1 ml transfection medium in a 1.6 ml microcentrifuge tube .
note : the vector ptagcfp - c - myc expresses the tagcfp - tagged c-myc.add 1.8 l of the transfection reagent to 0.1 ml transfection medium in another 1.6 ml microcentrifuge tube .
typically , prepare a master mix of diluted transfection reagent enough for 12 samples.incubate for 5 min , at room temperature.add the diluted 0.1 ml transfection reagent to the diluted 0.1 ml dnas .
spin the tubes for 5 sec in a microcentrifuge.incubate for 45 min , at room temperature . 
 
the next day , inside a biosafety cabinet , dilute 0.6 g ( total ) of the expression plasmid for tdtomato - tagged wild - type or motorless kif5b in the presence or absence of an expression plasmid for a cargo candidate protein ( ptagcfp - c - myc ) with 0.1 ml transfection medium in a 1.6 ml microcentrifuge tube .
note : the vector ptagcfp - c - myc expresses the tagcfp - tagged c - myc .
add 1.8 l of the transfection reagent to 0.1 ml transfection medium in another 1.6 ml microcentrifuge tube . typically , prepare a master mix of diluted transfection reagent enough for 12 samples .
washing of cells wash the seeded cells three times with phosphate buffered saline ( pbs ) during the incubation period for the formation of the transfection complex.replace the medium of the seeded cells in each well with 0.8 ml pre - warmed transfection medium .
wash the seeded cells three times with phosphate buffered saline ( pbs ) during the incubation period for the formation of the transfection complex . replace the medium of the seeded cells in each well with 0.8 ml pre - warmed transfection medium .
transfection of cells after incubation of 45 min , add 0.2 ml of the dna / transfection reagent complex ( prepared in step 2.3 ) dropwise to each well of the plate.rock the 6 well plate gently for 5 sec , before they are returned to the incubator at 37 c.after 6 - 8 hr , replace the medium with the complete dmem medium . 
 
after incubation of 45 min , add 0.2 ml of the dna / transfection reagent complex ( prepared in step 2.3 ) dropwise to each well of the plate .
rock the 6 well plate gently for 5 sec , before they are returned to the incubator at 37 c .
after 6 - 8 hr , replace the medium with the complete dmem medium . for live - cell fluorescence imaging 
 after an additional 16 hr of incubation at 37 c , add the dna intercalating stain hoechst 33342 to the medium to a final concentration of 1 m .
note : hoechst 33342 is a cell - permeable , dna intercalating dye that stains the cell nuclei.incubate the transfected cells for 10 min in the incubator at 37 c before examining them for the expression of fluorescent proteins by fluorescent microscopy using a 40x objective .
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively . note : if the background is high due to auto - fluorescence of the complete dmem medium , medium without phenol red is used . 
  after an additional 16 hr of incubation at 37 c , add the dna intercalating stain hoechst 33342 to the medium to a final concentration of 1 m . note : hoechst 33342 is a cell - permeable , dna intercalating dye that stains the cell nuclei . incubate the transfected cells for 10 min in the incubator at 37 c before examining them for the expression of fluorescent proteins by fluorescent microscopy using a 40x objective .
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively . note : if the background is high due to auto - fluorescence of the complete dmem medium , medium without phenol red is used .
preparation of paraformaldehyde solution weigh paraformaldehyde powder ( 4 g per 100 ml pbs ) and add it to pbs .
therefore , it is important to avoid contact with or inhalation of paraformaldehyde.add naoh to the solution ( 150 l 10 n naoh/100 ml).keep the solution at 37 to 42 c for 2 - 3 hr with occasional shaking.adjust ph to 7.0 by adding glacial acetic acid to the solution ( about 75 l/ 100 ml ) after the paraformaldehyde powder is dissolved . 
 
weigh paraformaldehyde powder ( 4 g per 100 ml pbs ) and add it to pbs .
keep the solution at 37 to 42 c for 2 - 3 hr with occasional shaking .
adjust ph to 7.0 by adding glacial acetic acid to the solution ( about 75 l/ 100 ml ) after the paraformaldehyde powder is dissolved .
target proteins staining after further incubation for 16 hr at 37 c , wash the transfected cells at room temperature once with pbs by swirling the six - well plate for 5 sec.fix the cells with 1 ml of freshly prepared , 4% paraformaldehyde solution per well .
incubate at room temperature for 30 min.wash the cells with pbs once by swirling for 5 sec the six - well plate .
discard the solution.incubate the cells with 1 ml of 0.1% triton - x 100 ( in pbs ) at room temperature for 30 min .
the detergent triton - x 100 will permeabilize the cell membrane to allow access of antibodies to their intracellular targets.wash the cells with pbs four times by swirling the six - well plate for 5 sec each time . discard the solution after each wash.incubate the cells with 1 ml of primary antibodies ( c - myc rabbit antibody or p53 mouse antibody ; 0.1 g / ml ) in a 10% fbs in pbs solution at room temperature by rocking for 4 hr.wash with pbs four times by swirling the six - well plate for 5 sec each time .
discard the solution after each wash.incubate with 1 ml of fluorescent dye - conjugated secondary antibodies ( alexa fluor 488-conjugated anti - rabbit or anti - mouse igg antibody ; 0.5 g / ml ) in 10% fbs in pbs at room temperature in the dark by rocking for 2 hr.wash with pbs four times by swirling the six - well plate for 5 sec each time . discard the solution after each wash . 
  after further incubation for 16 hr at 37 c , wash the transfected cells at room temperature once with pbs by swirling the six - well plate for 5 sec .
fix the cells with 1 ml of freshly prepared , 4% paraformaldehyde solution per well .
wash the cells with pbs once by swirling for 5 sec the six - well plate .
incubate the cells with 1 ml of 0.1% triton - x 100 ( in pbs ) at room temperature for 30 min .
the detergent triton - x 100 will permeabilize the cell membrane to allow access of antibodies to their intracellular targets .
wash the cells with pbs four times by swirling the six - well plate for 5 sec each time .
incubate the cells with 1 ml of primary antibodies ( c - myc rabbit antibody or p53 mouse antibody ; 0.1 g / ml ) in a 10% fbs in pbs solution at room temperature by rocking for 4 hr .
wash with pbs four times by swirling the six - well plate for 5 sec each time .
incubate with 1 ml of fluorescent dye - conjugated secondary antibodies ( alexa fluor 488-conjugated anti - rabbit or anti - mouse igg antibody ; 0.5 g / ml ) in 10% fbs in pbs at room temperature in the dark by rocking for 2 hr .
wash with pbs four times by swirling the six - well plate for 5 sec each time .
discard the solution after each wash . nuclear staining and mounting incubate the cells with the 1 ml of dna intercalating dye 4',6-diamidino-2-phenylindole ( dapi ; 0.5 g / ml ) in pbs at room temperature , in dark for 10 min .
note : dapi is a dna intercalating dye that stains the cell nuclei.apply 10 l of mounting solution onto each microscope slide .
mount each cover glass over the mounting solution on a microscope slide.incubate at room temperature in the dark overnight.seal the edges of the cover glasses with nail polish .
place in the dark inside a fume hood overnight to remove the nail polish fumes . 
 
incubate the cells with the 1 ml of dna intercalating dye 4',6-diamidino-2-phenylindole ( dapi ; 0.5 g / ml ) in pbs at room temperature , in dark for 10 min .
seal the edges of the cover glasses with nail polish . place in the dark inside a fume hood overnight to remove the nail polish fumes .
fluorescence microscopy 
 next day , examine the cells by fluorescent microscopy using a 40x objective .
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively . 
 
the filter sets for dapi , cfp , fitc and cy3 are ( ex350 nm/ em460 nm ) , ( ex436 nm/ em480 nm ) , ( ex470 nm/ em525 nm ) and ( ex545 nm/ em605 nm ) , respectively .
exogenously expressed wild - type kif5b appeared homogeneously in the cytoplasm , while c - myc appeared mainly in the nucleus ( figure 1a ) .
however , the motorless kif5b mutant formed aggregates in the cytoplasm ( figure 1a ) .
the percentage of cells expressing aggregated tagcfp - tagged c - myc in the cells expressing wild - type kif5b was low .
however , it was significantly higher when the motorless kif5b was co - expressed ( figure 1a ) .
the observed co - localization of mutant kif5b with c - myc ( figure 1 ) suggests that kif5b regulates the subcellular localization of c - myc and c - myc is a cargo of kinesin-1 .
negative controls in which the constructs were expressed alone are included in the lower panel of figure 1a .
the motorless kif5b also induced aggregation of the endogenous c - myc ( figure 1b ) and the transcription factor p53 ( figure 2 ) , indicating thatc - myc and p53 are both the cargos of kinesin-1 and kif5b regulates the subcellular localization of both endogenous proteins .
together with the results that the kinesin-1 inhibitor rose bengal lactone ( rbl ) induces formation of high molecular weight species for both c - myc and p53 , p53 is likely a cargo of kinesin-1 .
it is interesting to note that the nuclear transcription factor p53 , like c - myc , is also exported from the nucleus to the cytoplasm for proteasomal degradation .
the movement of transcription factors by kif5b appears to be specific because the expression of the motorless kif5b mutant did not affect the subcellular localization c - fos or cause it to aggregate ( figure 3 ) .
the above data demonstrate that the method employed in this publication allows for the high - specificity identification of kinesin-1 cargos .
figure 1 : expression of the motorless kif5b mutant induces c - myc aggregation in the cytoplasm .
( a ) hela cells were transfected with tdtomato - tagged wild - type ( wt ) kif5b ( red ) and tagcfp - tagged c - myc ( blue ) .
tdtomato - tagged wt kif5b appeared mainly in the cytoplasm , while tagcfp - tagged c - myc appeared mainly in the nucleus .
however , the tdtomato - tagged motorless kif5b mutant ( red ) formed filamentous or punctate aggregates in the cytoplasm .
percentages of cells ( % ) indicating aggregates of cfp - c - myc ( % ) are shown .
negative controls with expression of tdtomato - tagged kif5b proteins or tagcfp - tagged c - myc along with empty vectors ( v ) are shown in the lower panel .
( b ) similar results were obtained with the endogenous c - myc ( green ) when tdtomato - tagged wt or motorless kif5b proteins ( red ) were expressed .
the tdtomato - tagged motorless kif5b mutant formed aggregates in the cytoplasm and induced aggregation of the endogenous c - myc . both kinds of aggregates co - localized together in the cytoplasm ( orange ) .
figure 2 : expression of the motorless kif5b mutant induces endogenous p53 aggregation in the cytoplasm . in hela cells ,
exogenous tdtomato - tagged wild - type ( wt ) kif5b ( red ) appeared mainly in the cytoplasm , while the endogenous p53 ( green ) appeared mainly in the nucleus .
in contrast , the tdtomato - tagged motorless kif5b mutant ( red ) formed aggregates in the cytoplasm .
expression of the motorless kif5b induced the aggregation of p53 , resulting in the co - localization of kif5b and p53 in the cytoplasm ( yellow ) .
figure 3 : expression of the motorless kif5b mutant does not induce aggregation of c - fos in the cytoplasm .
tdtomato - tagged wild - type ( wt ) kif5b ( red ) appeared mainly in the cytoplasm of hela cells , while egfp - c - fos ( green ) appeared mainly in the nucleus . on the contrary , the tdtomato - tagged motorless kif5b mutant ( red ) formed aggregates in the cytoplasm .
table 1 : primer sequences for cloning the wild - type and motorless kif5b proteins . 

the method described utilizes the properties of the motorless kif5b mutant , which lacks the ability to move along the microtubules , but retains the ability to form dimers with the wild - type kif5b and , thereby , allow the tetrameric protein to interact with the same cargo proteins as the wild - type kif5b .
motorless kif5b , therefore , acts as a dominant negative mutant and forms mislocalized aggregates with its cargos .
this method is proven to identify the kinesin-1 cargo c - myc ( figure 1 ) . in this article , the same motorless kif5b
mutant was used to identify p53 as another potential cargo of kinesin-1 ( figure 2 ) .
furthermore , the specificity of the mutant is provided by the lack of effect of the mutant on the negative control protein c - fos ( figure 3 ) . in this protocol , the tdtomato - tagged wild - type or motorless kif5b protein is coexpressed with another fluorescent protein - tagged candidate cargo protein . in this case , live - cell fluorescence microscopy and imaging are performed .
alternatively , the tdtomato - tagged protein is expressed alone and the candidate cargo protein at its physiological levels is visualized by indirect immunofluorescence microscopy using specific antibodies .
if the background is high due to auto - fluorescence of the complete dmem medium , medium without phenol red is used .
therefore , it is critical to express sufficient amount of motorless kif5b mutant to form dimers with the wild - type counterpart to inhibit its function and to form aggregates . to address this issue , optimization of expression of the motorless mutant is essential .
the duration of incubation of hela cells with the dna/ transfection reagent was optimized in hela cells for expression of exogenous proteins and cell viability .
the incubation duration may have to be optimized for other cell lines . for magnifications between 4x to 40x ,
therefore , in this case , the protocol is inexpensive and convenient . for magnifications above 40x ,
cells are grown on cover glasses in the wells and , after staining , are mounted on microscope slides for examination under oil - immersion objectives .
observation of aggregates of the motorless kif5b protein is limited by the size of the cytoplasm .
however , it is relatively difficult to observe the aggregates in neuronal cells when the volume of the cytoplasm is small around the nuclei and in neurites .
the protocol is used to show the association between kif5b and its cargos in addition to the in vivo yeast two - hybrid and biochemical pull - down assays .
all these assays determine the association under different physical conditions and their results can complement each other .
moreover , the advantage of this fluorescence assay over other assays is that it can show the regulation of subcellular localization of the cargos by kif5b ( figures 1 and 2 ) .
the protocol is not limited to kif5b and can be used to identify cargos of other motor proteins , such as some other kinesin motors and some of the myosin motors used in intracellular transport of cargos .
these motor proteins also contain motor domains for movement along microtubules for kinesin motors and microfilaments for myosin motors , and coiled - coil segments for oligomerization .
therefore , a similar strategy can be applied to them by creating their motorless dominant negative mutants to identify their cargos .
 fluorescence microscopy is employed to identify kinesin-1 cargos . recently 
 , the heavy chain of kinesin-1 ( kif5b ) was shown to transport the nuclear transcription factor c - myc for proteosomal degradation in the cytoplasm . 
 the method described here involves the study of a motorless kif5b mutant for fluorescence microscopy . 
 the wild - type and motorless kif5b proteins are tagged with the fluorescent protein tdtomato . 
 the wild - type tdtomato - kif5b appears homogenously in the cytoplasm , while the motorless tdtomato - kif5b mutant forms aggregates in the cytoplasm . 
 aggregation of the motorless kif5b mutant induces aggregation of its cargo c - myc in the cytoplasm . 
 hence , this method provides a visual means to identify the cargos of kinesin-1 . a similar strategy can be utilized to identify cargos of other motor proteins . 
Introduction
Protocol
1. Cloning of the tdTomato-tagged Wild-type and Motorless KIF5B Proteins
2. Immunofluorescence Microscopy
3. For Indirect Immunofluorescence Studies
Representative Results
Discussion
Disclosures