FIG. 7.
Analysis of synchronized p16-minus Jurkat leukemic T cells. (A) Asynchronous Jurkat T cells were synchronized by centrifugal elutriation. Early-G1, late-G1, and S-phase cellular fractions were analyzed for pRB phosphorylation status (immunoblot analysis), cyclin E-Cdk2 and cyclin D-Cdk4/6 kinase activities, and DHFR mRNA levels (RT-PCR analysis). αpRB blot, anti-pRB blotting; Hyper-PO4, hyperphosphorylated; Hypo-PO4, hypophosphorylated; arb., arbitrary. (B) Early-G1-phase elutriated Jurkat cellular fractions from the above-described experiment were immunoprecipitated with anti-E2F1 or control anti-IgG (αM) antibodies, followed by anti-pRB immunoblot analysis. pRB-E2F1 complexes remained assembled in early-G1-phase cells. IP, immunoprecipitates.