Cyclin D3 expression is dependent on Notch signaling in CD4⁺ T cells. (A) Whole cell lysates of CD4⁺ T cells were analyzed by immunoblotting for cyclin D3 or Notch1^IC expression. CD4⁺ T cells were stimulated with 1 μg/mL anti-CD3ϵ and 1 μg/mL anti-CD28 for indicated time periods (0, 3, 6, and 12 hours) in the presence of GSI (IL-CHO, 50 μm) or DMSO (0.1%). (B) Later time points (24, 48, and 72 hours) after 1 μg/mL anti-CD3ϵ and 1 μg/mL anti-CD28 stimulation were analyzed for differences in cyclin D3 and Notch1^IC expression with or without GSI treatment. (C) Whole cell lysates from stimulated CD4⁺ T cells (for 24, 48, and 72 hours) that were isolated from C57BL/6 mice treated in vivo with GSI for 14 days were analyzed for cyclin D3 and Notch1^IC. GAPDH was used as a loading control. LY indicates GSI in rodent chow; and C, control rodent chow.