Figure 5.

Phosphorylation of Ser 149 prevents G3BP self-association. (A) Whole-cell extracts prepared from Cos cells transfected with GFP, GFP-G3BP wild type, and phosphorylation mutants GFP-S149A, GFP-S149E, GFP-S232A, and GFP-S232E were immunoprecipitated with anti-GFP antibodies. Proteins were revealed by immunoblot analysis using anti-G3BP antibody. S, supernatant; IP, immunoprecipitation. (B) Glutaraldehyde cross-linking analysis of purified G3BP phosphorylation mutants S149A (lanes 1–6) and S149E (lanes 7–11). Proteins (0.2 μg each) were incubated for the indicated time with glutaraldehyde (G.A.), and were then analyzed by Western blotting with an anti-G3BP. Asterisks correspond to proteolytic fragments of G3BP-GFP that can be detected by anti-GFP antibodies, whereas the band at level of G3BP is only seen with antiG3BP antibody.