TIF-IA as a cofactor for Ebp1-regulated PCNA transcription. (A) Effects of Ebp1 depletion on PCNA expression and proliferation in Jurkat T cells. Cells were transfected with SCR or two concentrations of siEbp1 for 36 hours. Western blot with the indicated antibodies (left), PCNA mRNA level (middle), and MTS and colony forming assays (right). (B) Expression of Ebp1 in Ebp1^+/+ and Ebp1^−/− MEF cells. Western blot was probed with the indicated antibodies (left); PCNA mRNA level (middle) and MTS assay (right). (C) Interaction of Ebp1 and TIF-IA. T-cell lysate was incubated with anti-Ebp1 or TIF-IA antibody and western blots were probed with the antibodies shown (left) and Jurkat T cells were co-transfected with Myc vector alone or Myc–TIF-IA and GFP–Ebp1 (right). Cell lysate was incubated with anti-Myc antibody and the western blot probed with anti-GFP antibody. (D) Effect of T-cell activation with anti-CD3/CD28 on the interaction of Ebp1 and TIF-IA, PCNA expression, and proliferation. Lysate from resting or activated T cells was incubated with anti-Ebp1 antibody and the western blots were probed with the indicated antibodies (left), PCNA mRNA expression (middle), and MTS assay (right). (E) Effect of depletion of Ebp1, TIF-IA, or both in primary activated T cells on PCNA expression (left and middle) and cell proliferation (right). (F) Effect of Ebp1 overexpression in Jurkat T cells in the absence or presence of TIF-IA on PCNA expression (left and middle) and proliferation (right). The cells were pretreated with SCR or siTIF-IA for 24 hours and continuously transfected with GFP Ebp1 for an additional 24 hours.