also prepared for analysis of gsk 3 8 activity muscle homogenates 6 u03bcg in 25 u03bcl of homogenization buffer were mixed with 12 5 u03bcl of 1 11 mm substrate peptide b 2b ps anaspec inc san jose calif based on the primed elf2b consensus sequence for gsk 3 phosphorylation biotin rraaeeldsragpspql oh seq id no 2 and the reaction was started upon addition of 12 5 u03bcl 4 u00d7 atp mix 200 mm tris hcl ph 7 4 50 mm mgcl 2 8 mm dtt 0 4 mm atp and 0 08 u03bcci u03bcl 33 p gamma atp 3000 ci mmol perkin elmer life sciences boston mass neg602h incubations were carried out at 30 u00b0 c 9 u03bcl aliquots of reaction mix were removed at 5 10 20 and 30 min and the reaction was stopped by mixing with 9 u03bcl of 1 n hcl on ice stopped reaction mix 10 u03bcl was spotted onto streptavidin coated paper squares sam2 membranes promega madison wis squares were washed 1 u00d7 30 sec and 3 u00d7 2 min in 2 m nacl followed by 4 u00d7 2 min in 2 m nacl 1 phosphoric acid and then 2 u00d7 in distilled water and once in ethanol the squares were dried and counted on a wallac scintillation counter perkin elmer life sciences boston mass the dpm 39 s were converted to pmoles product formed and graphed as a function of time the slopes were determined by linear fit using graphpad prism u00ae graphpad software inc san diego calif and are equivalent to mu gsk 3 u03b2 activity pmoles product formed per minute as shown in fig3.