Caprin1 and USP10 binding to G3BP is mutually exclusive and regulates SG formation. (A) COS7 cells were transiently transfected with the indicated GFP (green) or Cherry (red)-tagged constructs and stained for endogenous eIF4G (1 and 2, red; 3, green) and/or eIF3b (1–6, blue). In 6, the cells were treated with 200 µM AS and then fixed and stained. Bar, 10 µm. Insets zoomed 2.5× with separated colors. (B) COS7 were transiently transfected with the indicated constructs, lysed and immunoprecipitated. Lysates and IPs were resolved using SDS-PAGE and subjected to Western blotting for the indicated proteins. Lower panels, duplicate samples probed for GFP to confirm IP efficiency. M_r (kD) are shown. (C) Endogenous G3BP1 from U2OS-WT cell lysates was immunoprecipitated, and the bound complexes were incubated with the indicated amounts of USP10_8–25WT or mutant USP10_8–25F10A peptides. Bound and released material was subjected to Western blotting for endogenous G3BP1, USP10, or Caprin1. (D) Quantification of displaced Caprin1 and USP10. Western blots from samples in C were quantified using densitometry. The ratio of each protein relative to G3BP1 was determined. Data shown are mean ± SEM and are analyzed using the unpaired t test. *, P < 0.05; n = 3. (E) Purified recombinant His-G3BP was mixed with biotinylated USP10_8–25WT or mutant USP10_8–25F10A peptides and bound to streptavidin (SA) beads. Beads were then washed and incubated with the indicated amounts of purified recombinant His-Caprin1. Bound material was subjected to Western blotting for G3BP1 and Caprin1. n = 3. M_r (kD) are shown.