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. 2022 Dec 27;42(3):e111802. doi: 10.15252/embj.2022111802

Figure EV4. Inhibition of SUMO‐targeted ubiquitylation partially restores UPS activity in thermally stressed G3BP1/2 knockout cells.

Figure EV4

  1. U2OS G3BP1/2 knockout cells expressing Ub‐YFP were transfected with control siRNA or RNF4 siRNA. Cell lysates were analyzed by immunoblot with RNF4 and Actin antibodies.
  2. Parental U2OS and G3BP1/2 KO cells expressing Ub‐YFP were transfected with control siRNA or RNF4 siRNA. Cell lysates were analyzed by immunoblot with RNF4 and Actin antibodies.
  3. Fluorescence micrographs of parental U2OS cells stably expressing Ub‐YFP, which had been transfected with control or RNF4 siRNA. Cells were either left untreated (− heat shock), or exposed to 43°C for 30 min and followed for 4 h after heat shock (Recovery). Scale bar is 10 μm.
  4. Fluorescence micrographs of U2OS G3BP1/2 KO cells stably expressing Ub‐YFP, which had been transfected with control or RNF4 siRNA. Cells were either left untreated (− heat shock), or exposed to 43°C for 30 min and followed for 4 h after heat shock (Recovery). Scale bar is 10 μm.
  5. Immunoblotting for PML in control (siControl) and PML‐depleted (siPML) parental and G3BP1/2 KO U2OS cells.
  6. Quantification of mean cellular YFP fluorescence intensities in U2OS expressing Ub‐YFP with and without PML siRNA transfection. The YFP fluorescence is normalized to untreated control cells. The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n = 3 independent experiments, > 1,000 cells analyzed per condition, Kruskal‐Wallis test, *P < 0.05, **P < 0.01).
  7. Immunoblot of ataxin‐1 in parental and G3BP1/2 KO U2OS cells transfected with FLAGUb‐Ataxin‐1‐82Q.
  8. Immunoblot of FLAGUbiquitin in parental and G3BP1/2 KO U2OS cells transfected with FLAGUb‐Ataxin‐1‐82Q.