Fig. 3: sERr is attenuated by translation inhibition and involves the generation of intermolecular disulfide bonds formation with PDI proteins.

a HepG2 WT cells were treated with either DMSO or Tg (0.2 μg/ml) plus GSK414 (0.5 μM) in the presence of different concentrations of cycloheximide (CHX) for 16 h followed by immunoblotting against c-MET, EGFR, and p97. The quantitative ratio between ER-arrested c-MET to cell-surface located c-MET is shown as pro-MET/mature MET for each treatment. Experiment was performed twice with a similar outcome. b Flow cytometry analysis of EGFR and c-Met of HepG2 WT cells following 16 h treatment either with DMSO in presence/absence of CHX (600 ng/ml) or with Tg(0.2 μg/ml) plus GSK414(0.5 μM) in presence/absence of CHX(600 ng/ml). c Reducing and nonreducing immunoblotting for KIT in Mel526 cells (left) and c-MET in HepG2 cells (right) treated with DMSO, Tg, GSK414 alone or with Tg/GSK414 combination (typical results of three repetitions). d Reducing and nonreducing immunoblotting for ERp44 and PDI of HepG2 cells treated either with DMSO, Tg, GSK414, or both for 16 h (typical results of three repetitions). e Mel526 KIT KO cells were generated by CRISPR/Cas9 gene editing (lane 1 is WT cells where lane 2 is a KIT KO). The fusion protein KIT-3xFLAG was then stably expressed in the KO cells (lane 3). ER retention of the fusion protein was validated by treatment with Tg + GSK414 (lane 4). Mel526 KIT-3xFLAG expressing cells were treated either with DMSO or with Tg/GSK414 for 16 h. f Total lysate or anti-FLAG immunoprecipitates were immunoblotted for KIT, PDI, and ERp44 with ponceau-s staining(right) (typical results of three repetitions).