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. Author manuscript; available in PMC: 2019 May 9.
Published in final edited form as: Cell Rep. 2019 Apr 30;27(5):1356–1363.e3. doi: 10.1016/j.celrep.2019.04.015

Figure 4. Unconjugated Ubiquitin Localizes to SGs in a UAE-Independent Manner.

Figure 4.

(A–E) Immunofluorescence staining of HeLa cells treated with NaAsO2 (250 μM) for 0’, 45’, or 120’ prior to fixation (A) or pretreated with DMSO or UbE1i (1 μ M) for 90’, followed by treatment with NaAsO2 (250 mM) for 120’ prior to fixation (B–E). Cells were stained with antibodies against G3BP1 (A–E), pan-ubiquitin (A), polyubiquitin (B and D), centrosome marker pericentrin (D), proteasome subunits (D), VCP/p97 (E), SQSTM/p62 (E), HDAC6 (E), UBAP2L (E), or TRIM 25 (E). (C) Cells stably expressing either mCherry-tagged wild-type ubiquitin (mCh-Ub-WT) or mCherry-tagged ubiquitin, in which all internal lysine residues were mutated to arginine and the C-terminal diglycine residues were removed (mCh-Ub-K0DGG [K0dGG]) were stained with antibodies against G3BP1. G3BP1-positive SGs are indicated by arrows (both red and white). G3BP1-negative perinuclear foci are indicated by solid yellow arrowheads. G3BP1-negative punctate signal for VCP/p97 and SQSTM/p62 is indicated by blue open arrowheads. Images from Ub-E1i-treated cells in all panels were taken at the same exposure time and acquisition settings in the ubiquitin channel as those for images from DMSO-treated cells. Nuclei were stained using DAPI. Scale bars, 20 μ m in all panels.

See also Figure S4.