serum 1 penicillin and streptomycin 10 mg ml insulin t47d were grown in dmem with 10 fetal bovine serum 0 2 units ml insulin 1 penicillin streptomycin and 0 15 sodium bicarbonate expression plasmid encoding full length brca1 in pcdna3 psg5 er u03b1 and ere tk luciferase reporter pcdna3 ubc9 pcdna3 ubc9 c93s both v5 tagged and pcdna3 6 u00d7 his sumo 1 and pgex ubc9 were generously provided by dr michael erdos national human genome research institute bethesda md usa prof m g parker london u k prof ron hay and dr ellis jaffrey university of dundee u k and dr michael j mutanis john hopkins university respectively the pcdna3 brca1a amp pcdna3 brca1b plasmids have been described previously wang h shao n ding o m cui j reddy e s p rao v n brca1 proteins are transported to the nucleus in the absence of serum and splice variants brca1a brca1b are tyrosine phosphoprotein that associate with e2f cyclins and cyclin dependent kinases the cdnas for brca1 1 182 were constructed by pcr cloning and sub cloning into the pcmv tag 2 vector stratagene la jolie calif usa according to the directions this vector allows expression of brca1 protein with an n terminal flag sequence brca1 1a 1b brca1 1 182 point mutants 1 and 4 see fig1 b and text were constructed using quick change site directed mutagenesis kit stratagene gst ubc9 and gst er u03b1 337 379 were expressed and purified from e coli as described previously wang h shao n ding o m cui j reddy e s p rao v n brca1 proteins are transported to the nucleus in.