Figure 6

Acetylation controls the ability of Tax to stimulate phosphorylation of pRb by CDK4-cyclin D3-p21^CIPcomplexes. CHO cells were transfected with vectors expressing CDK4, cyclin D3 and either 0.2 or 2 μg of vector expressing p21 with or without a vector expressing Tax-HA (A), or 2 μg of vector expressing p21 with vectors expressing WT or mutant K₃₄₆R or K₃₄₆Q Tax-HA (B), or 2 μg of vector expressing p21 and WT Tax-HA and 1 or 2 μg of vector expressing HDAC7-Flag (C). Total lysates were immunoprecipitated with the anti-cyclin D3 mAb and both the total lysates and the immunoprecipitated complexes (IP anti-cyclin D3) were analyzed by Western Blotting using antibodies shown on the left side of the lanes. The immunoprecipitated complexes were also analyzed using an in vitro pRb phosphorylation assay. The diagrams represent the relative CDK4 specific activity obtained by quantitation of the intensity of the pRb phosphorylated species on the blots reported to equal amount of CDK4 in the complexes. One representative experiment is presented in panel A. Error bars were calculated for two independent experiments in panels B and C.