FIG. 2.

ERF is phosphorylated in the nucleus. (A) Ref cells were subjected to serum arrest for 2 h and consequent serum stimulation for the indicated times. The localization of the phosphorylated ERF was determined by indirect immunofluorescence using the P3-4 phospho-specific anti-ERF antibody (green), and that of total ERF was determined with the S17S anti-ERF antibody (red). Nuclei ware stained blue by TO-PRO-3 (left). At the same time points, activated Erks were detected with the phospho-specific anti-Erk monoclonal antibody MAPK-YT (green). Nuclei ware stained blue by TO-PRO-3 (right). Red and blue color colocalization shows as magenta, green and blue colocalization shows as cyan, green and red colocalization shows as yellow, and colocalization of all three shows as white. (B) Under the same conditions as in panel A, total cell extract was analyzed by immunoblotting for total and phosphorylated ERF and Erks, as indicated. The S17S, P3-4, P7, MAPK-YT, and anti-Erk specific polyclonal antibodies were used to determine total ERF, ERF phosphorylated at S246 and S251, ERF phosphorylated at T526, phosphorylated Erks, and total Erks, respectively. FBS, fetal bovine serum. (C) Ref cells were deprived of serum for 1 h and then induced with serum. At the indicated times cells were harvested and separated into nuclear and cytoplasmic fractions. The amount of the phosphorylated ERF protein in each fraction was determined by immunoblotting. Both the P3-4 (top) and the P7 (bottom) phospho-specific anti-ERF antibodies were used. (D) Ref cells were serum arrested for 2 h and then treated with the indicated amounts of epidermal growth factor for 5 min. Total cell extracts were analyzed for total and phosphorylated ERF and Erks, as for panel B.