FIG 4 .

G3BP1 dimerization is not required for PKR activation. (A) The indicated GFP alone, GFP-G3BP1, the NTF2 domain (amino acids 1 to 135), the GFP-G3BP1 F33W mutant, GFP-G3BP1 Δ1-11aa (lacking the first 11 amino acids of G3BP1), or GFP-G3BP2a was transfected into HeLa cells and precipitated with anti-GFP Sepharose. Precipitated material was analyzed by Western blotting against GFP, Caprin1, total PKR, P-PKR, and endogenous G3BP1. The arrow beside the endogenous G3BP1 blot indicates the correct band, while the asterisk highlights an unknown product originating from expression of the GFP-G3BP1 Δ1-11aa protein. ImageJ was used to quantify induction of P-PKR in the input, total PKR immunoprecipitated, and endogenous G3BP1 precipitated with the indicated GFP-tagged proteins. Band intensity for GFP alone was set to 1 in both cases. Nonessential lanes were removed from the gels in panel A (indicated by white space). (B) HeLa cells were transfected with GFP alone or G3BP1-GFP (green), and cells were stained with antibodies against t446 P-PKR (red). Untransfected (white “U”) and transfected (yellow “T”) cells are indicated. Results for all panels are representative of experiments repeated in triplicate.