Fig. 5: ER and proteotoxic stresses attenuate the actions of UBE3C on VPS34 by altering UBE3C partners.

a Immunoprecipitation analysis of the interaction between UBE3C and VPS34 in 293T cells treated with 500 μM thapsigargin (Tg), 10 μg/ml tunicamycin (Tu), 10 μM MG132, 10 μM 17-DMAG, 10 μM 17-AAG, 10 μg/ml puromycin (Puro) or starved (EBSS) for 30 min. To ensure a fair comparison, the amounts of immunoprecipitates loaded for Western blot were adjusted to allow an equal level of VPS34 appearing in treated and untreated cells. b, c Analysis of VPS34 ubiquitination in 293 T cells transfected with indicated constructs and treated with indicated agents for 1 h. d Western blot analysis of VPS34 levels in 293T cells treated with indicated agents for 1 h. e, g, i Confocal analysis of the colocalization of TagRFP-ATG16 with GFP-TRABID or GFP-UBE3C in transfected HeLa cells cultured in full medium (e) or treated with tunicamycin (g) or puromycin (i) for 1 h. Bar, 10 μm. The amount of TagRFP-ATG16 plasmid for transfected was adjusted to make a comparable TagRFP-ATG16 puncta number in UBE3C- and TRABID-expressing cells. f, h, j Quantitative data for e, g, and i, respectively. The percentage of GFP-TRABID or GFP-UBE3C puncta colocalizing with TagRFP-ATG16 puncta was analyzed and plotted. k Quantitative data for the absolute numbers of GFP-TRABID/TagRFP-ATG16 and GFP-UBE3C/TagRFP-ATG16 double-positive dots in cells treated with indicated agents. Data in f, h, j, and k are mean ± SD (n = 3 independent experiments, and 5 cells per group per experiment were counted). P-values are determined by unpaired two-sided t-test. l Immunoprecipitation analysis of the interaction between UBE3C and proteasome subunit S5A in 293T cells treated with indicated agents for 30 min. Source data are provided as a Source Data file.