Fig. 6: Enforced binding of UBE3C to VPS34 under ER or proteotoxic stress compromises proteostasis and ER quality control. | Nature Communications

Fig. 6: Enforced binding of UBE3C to VPS34 under ER or proteotoxic stress compromises proteostasis and ER quality control.

From: VPS34 K29/K48 branched ubiquitination governed by UBE3C and TRABID regulates autophagy, proteostasis and liver metabolism

Fig. 6: Enforced binding of UBE3C to VPS34 under ER or proteotoxic stress compromises proteostasis and ER quality control.The alternative text for this image may have been generated using AI.

a Schematic representation of the experimental design to induce UBE3C and VPS34 association in targeting cells but not in control cells. b 293T UBE3C knockout (KO)-derived targeting (T) or control cells (C) were treated with 10 μg/ml puromycin or tunicamycin together with or without 0.5 μM rapalog for 2 h and analyzed by Western blot. c Immunofluorescence analysis of 293T UBE3C KO-derived targeting or control cells treated with puromycin and/or rapalog for 2 h. Representative images are on the left and quantitative data are on the right. Boxed regions are enlarged. Bar, 10 μm. Data are mean ± SD (n = 3 independent experiments, and 30 cells per group per experiment were counted). P-values are determined by two-way ANOVA with Tukey’s post hoc test. d 293T UBE3C KO-derived targeting or control cells were treated as the scheme outlined and analyzed by immunostaining for ubiquitin/p62 double-positive aggregates. Representative images are on the left and quantitative data are on the right. Arrowheads indicate the double-positive dots. Bar, 10 μm. Data are mean ± SD (n = 3 independent experiments, and 30 cells per group per experiment were counted). P-values are determined by two-way ANOVA with Tukey’s post hoc test. e Confocal analysis of 293T UBE3C KO-derived targeting or control cells transfected with ssRFP-GFP-KDEL reporter and treated with tunicamycin and/or rapalog for 6 h. Representative images are on the left and quantitative data are on the right. Bar, 10 μm. Data are mean ± SD (n = 3 independent experiments, and 30 cells per group per experiment were counted). P-values are determined by two-way ANOVA with Tukey’s post hoc test. f Western blot analysis of cells as in e treated with tunicamycin, rapalog, and/or bafilomycin A1 for 6 h. g, h 293T UBE3C KO-derived targeting or control cells as in b were treated with puromycin (g) or tunicamycin (h) together with rapalog for 3 h (g) or 6 h (h) and analyzed for apoptosis. Data are mean ± SD (n = 3 independent experiments). P-values are determined by two-way ANOVA with Tukey’s post hoc test; ns, not significant. Source data are provided as a Source Data file.

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