IP R Cyclin E. K-Ras V12. c-Myc. WT p27-/- CK- WT p27-/- CK- WT p27-/- CK-. M P-HH1. IP R CDK2. K-Ras V12. c-Myc. WT p27-/- CK- WT p27-/- CK- WT p27-/- CK-. 32 P-HH1. IP R CDK1. K-Ras V12. c-Myc. - PowerPoint PPT Presentation
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 g of proteins) or tumors lysates (120 g of proteins) and subjected to in vitro kinase assays. Briefly, immunprecipitates were incubated in 20 l of CDK kinase buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2, 1 mM dithiothreitol, 10 mM -glycerophosphate, 1 mM NaF, 2.5 mM EGTA, 0.1mM sodium orthovanadate) containing 2 g of histone H1 and 200 M ATP. For 20 min at 30C. 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 l of 32P-ATP, gel slabs were dried and directly exposed on film.