wt p27-/- ck- wt p27-/- ck- wt p27-/- ck-
Post on 07-Feb-2016
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WT p27-/- CK- WT p27-/- CK- WT p27-/- CK-
K-Ras V12 c-Myc
IP Rα CDK2
32P-HH1
IP Rα Cyclin E
Mα P-HH1
WT p27-/- CK- WT p27-/- CK- WT p27-/- CK-
K-Ras V12 c-Myc
IP Rα CDK1
WT p27-/- CK- WT p27-/- CK- WT p27-/- CK-
WT
Tumors – IP Rα CDK2
p27-/- CK-
Mα P-HH1
Mα P-HH1
K-Ras V12 c-Myc
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.
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