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. 1999 Apr 27;96(9):4844–4849. doi: 10.1073/pnas.96.9.4844

Figure 4.

Figure 4

GST-Smad3 and GST-Smad4 associate with an inducibly phosphorylated form of endogenous cJun. (A) HaCaT cells were treated with 100 pM TGFβ1 for 15, 30, or 60 min and lysed. GST-Smad3 and GST-Smad4 were incubated with 450 μg of whole-cell HaCaT lysates, and the binding reactions were analyzed by Western blot by using a cJun specific monoclonal antibody raised against a phosphopeptide containing phosphorylated Ser-63 of cJun, KM-1 from Santa Cruz. The lower row shows 60 μg total lysate blotted with the same antibody as the upper rows. (B) Nuclear lysates (60 μg) from HaCaT cells treated as in A were analyzed with the KM-1 antibody. This blot was stripped and reprobed with antibody no. 9162 from NEB raised against a fusion protein containing the unphosphorylated N-terminal portion of cJun. (C) HaCaT cells were treated with 100 pM TGFβ1 or 0.5 M sorbitol for 15 min, lysed in the absence or presence of phosphatase inhibitors, and treated with or without potato acid phosphatase and calf intestinal phosphatase. The reactions were analyzed by SDS/PAGE and Western blotting. The upper row shows 60 μg of each lysate blotted with the KM-1 antibody. This blot was stripped and reprobed with no. 9162 antibody, shown in the middle row. This blot was stripped again and reprobed with no. 06–828 antibody (Upstate Biotechnology). The bands in the KM-1 blot align with the upper bands in the no. 9162 antibody and the no. 06–828 antibody blots.