Fig. 2. 14-3-3τ interacts with Ser³¹-phosphorylated E2F1.

A, Western blot analysis of E2F1 in HEK293 cells transfected with wild type (wt) E2F1 or S31A-E2F1 and treated with NCS (300 ng/ml) for the indicated time periods. β-actin immunoblotting (IB) was performed to ensure equal protein loading. B, 14-3-3τ interacts with ATM-phosphorylated E2F1. GST or GST-14-3-3τ was incubated with the cellular lysates from NCS-treated cells as described in A and pulled down with glutathione-Sepharose. The amounts of input lysates had been adjusted so that each input contained roughly equal levels of E2F1 (bottom). C, 14-3-3τ interacts with an E2F1 phosphopeptide. GST or GST-14-3-3τ was incubated with a biotin-labeled nonphosphorylated peptide (nP) derived from E2F1 sequences or a Ser³¹-phosphorylated peptide (pP) and pulled down with streptavidin-Sepharose (top) or glutathione-Sepharose (bottom) followed by GST immunoblotting. D, binding of 14-3-3τ with E2F1 peptides was tested by ELISA as described under “Experimental Procedures.” The data shown are the mean ± S.D. of triplicate experiments.