G3BP1-S99P exhibits reduced expression and increased ubiquitination, and recruits sequestosome into SGs. (A–D) COS7 cells expressing GFP-G3BPs (green), stained for sequestosome-1 (red) and eIF3b (blue). Zooms 2.75× below each panel, with colors separated (gray presents blue). Bar, 25 µm. (E) GFP-G3BPs transfected into COS7 cells, immunoprecipitated with GFP-TRAP, and blotted. (F) GFP-G3BP1s expressed in ΔΔG3BP1/2 U2OS cells, immunoprecipitated with GFP-TRAP, and blotted. (G) Quantification of Caprin1, USP10, and G3BP1 from F, showing fold change relative to G3BP1-WT (mean ± SEM, n = 8). (H) MS analysis of endogenous G3BP1-S149 and G3BP1-S232 phosphorylation following mock or 1-h SA (500 µM) treatment (mean ± SEM, n = 4). (I) MS analysis of stably expressed GFP-G3BP1-S149 and GFP-G3BP1-S232 phosphorylation in ΔΔG3BP1/2 U2OS cells during mock or 1-h SA (500 µM) treatment (mean ± SEM,n = 6).