Enhanced association of cdk2 with p21^Cip1/WAF1 occurs in EGF-stimulated HUVEC treated with PF4. (A) Immunoprecipitated cdk2, from HUVEC lysate, collected 15 hours after EGF stimulation in the absence (−) or presence (+) of 2 μg/mL PF4, was assayed with GST-pRB in the presence of [γ³²P]ATP/ATP as described in Materials and Methods. Phosphorylated GST-pRb was then analyzed by SDS-PAGE and autoradiography and was quantified with an AMBIS scanner. (B) cdk2 levels in aliquots of the samples used to generate the results shown in panel (A), were determined by SDS-PAGE and immunoblotting. (C) The quantity of cyclin E immunoprecipitated with cdk2 was determined by SDS-PAGE followed by immunoblot analysis. (D) p21^Cip1/WAF1associated with cdk2 was similarly assayed. The results shown are representative of three independent experiments.