Figure 3.

Pocket protein–dependent effects of APOE on cyclin A gene transcription. (a) Cyclin A promoter activity in early-passage aortic SMCs was determined as described in Methods. Promoter activity is plotted as fold stimulation relative to the FBS-treated cells in the absence of E7. Results show the mean ± SEM, n = 3, *P < 0.001 as compared with transfected cells stimulated with FBS alone. (b) Early-passage aortic SMCs were transiently cotransfected with the E2F-CAT plasmid and either the E7 expression vector (+E7) or empty vector (–E7). After serum starvation, the quiescent transfectants were directly stimulated with 10% FBS for 18 hours in the absence (control) or presence of 2 μM APOE. CAT enzyme levels were measured as described in Methods. Results show the mean ± SEM, n = 2, *P < 0.001 as compared with transfected cells stimulated with FBS alone (control; –E7). (c) Quiescent mouse aortic SMCs were serum stimulated in the absence or presence of 2 μM APOE for 18 hours. Collected cells were lysed and analyzed by immunoblotting for pRb, p107, and cdk4. The hypophosphorylated and hyperphosphorylated forms of pRb and p107 are shown by lower and upper arrows, respectively. (d) Quiescent A10 SMCs were serum stimulated in the absence or presence of 2 μM APOE for 15 hours, collected, lysed, and analyzed by immunoblotting for cyclin E. Cell lysates were incubated with anti–cyclin E, and the immunoprecipitates were used to assess in vitro kinase activity or were analyzed by immunoblotting for associated cdk2.