Tumor CDK Activity Comparison in p27-/- and p27CK-/- MEFs

1. IP Rα Cyclin E K-Ras V12 c-Myc WT p27-/- CK- WT p27-/- CK- WT p27-/- CK- Mα P-HH1 IP Rα CDK2 K-Ras V12 c-Myc WT p27-/- CK- WT p27-/- CK- WT p27-/- CK- 32P-HH1 IP Rα CDK1 K-Ras V12 c-Myc WT p27-/- CK- WT p27-/- CK- WT p27-/- CK- Mα P-HH1 Tumors – IP Rα CDK2 WT p27-/- CK- Mα P-HH1 Supplemental Figure 5: CDK activity is similar in p27-/- and p27CK- MEFs and tumors. The indicated Cyclin or CDK were immunoprecipitated in exponentially growing MEF (500 μg of proteins) or tumors lysates (120 μg of proteins) and subjected to in vitro kinase assays. Briefly, immunprecipitates were incubated in 20 μl of CDK kinase buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2, 1 mM dithiothreitol, 10 mM β-glycerophosphate, 1 mM NaF, 2.5 mM EGTA, 0.1mM sodium orthovanadate) containing 2 μg of histone H1 and 200 μM ATP. For 20 min at 30°C. Reaction was ended by adding 4x loading buffer and boiling. Samples were resolved on SDS-PAGE, proteins were transferred on PVDF membranes and phosphorylated histone H1 was detected using a monoclonal anti phospho-histone H1 antibody (Millipore). For radioactive kinase assays, cold ATP was replaced with 0.5 μl of γ32P-ATP, gel slabs were dried and directly exposed on film.