CDK4 and CDK6 expression and pRb phosphorylation is dependent on Notch signaling in CD4⁺ T cells. (A) Whole cell lysate of CD4⁺ T cells that were stimulated as described before for indicated time points (0, 3, 6, and 12 hours) in the presence (in vitro) of GSI or DMSO were analyzed by immunoblotting for differences in CDK4 and CDK6 expression. (B) Whole cell lysates of CD4⁺ T cells that had been stimulated, as described, for indicated time points (24, 48, and 72 hours) in the presence (in vitro) of GSI or DMSO were analyzed by immunoblotting for differences in CDK4 and CDK6 expression. Changes in temporal expression of CDK4 and CDK6 after stimulation are represented graphically in lower panels. (C) Nuclear lysates from in vitro GSI- or DMSO-treated CD4⁺ T cells that were stimulated for 24, 48, and 72 hours were analyzed by immunoblotting for differences in pRb phosphorylation at residues Ser780 (top panel) and Ser795 (bottom panel). The nuclear protein PARP was used as a loading control.