Figure 5. Active ubiquitylation is required for normal stress granule clearance.

(A) HeLa cells were pre-treated with TAK-243 (1 μM, 1 h), followed by the addition of arsenite (250 μM) for 1 h in the continued presence of TAK-243. Subsequently, the cells were either fixed immediately or washed and allowed to recover under normal growth conditions over the indicated time course. SGs and Ub conjugates were visualized by confocal immunofluorescence microscopy using anti-G3BP and FK2 antibodies, respectively. Representative cells are magnified in the inset. Scale bar, 10 μm. (A, B) Immunoblot of whole-cell extracts from HeLa cells treated as in (A), using antibodies recognizing all forms of Ub (pan-Ub) or tubulin as loading control. (A, C) Quantification of cells with SGs in (A); shown is the mean ± SEM; n = 3 with ≥50 cells per replicate and condition; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; t test.