Figure 5.

The Δ48 cyclin E splice variant is functionally active. 76N-E6 cells were transiently transfected with cyclin EL–FLAG, Δ48–FLAG or the vector backbone (pCDNA3.1) using the FuGENE reagent. Twenty-four hours following transfection cells were harvested and cell lysates were prepared and subjected to (A) western blot analysis, (B) FLAG kinase analysis and (C) FLAG immune complex formation. For western blot analysis 50 µg protein extract from each condition were analyzed by western blotting with the anti-FLAG antibody. The blot was developed with chemiluminescence reagents. For FLAG kinase activity equal amounts of protein (250 µg) from cell lysates were prepared for each condition and immunoprecipitated with anti-FLAG antibody (polyclonal) coupled to protein A beads, using histone H1 or GST-Rb as substrate. For each condition we show the resulting autoradiogram of the histone H1 and GST-Rb SDS–PAGE and the quantitation of the histone H1 and GST-Rb associated kinase activities by Cerenkov counting. For immunoprecipitation followed by western blot analysis equal amounts of protein (250 µg) from cell lysates prepared for each condition were immunoprecipitated with anti-FLAG (polyclonal) antibody coupled to protein A beads and the immunoprecipitates were subjected to western blot analysis with CDK2 and p21 antibodies.