Fig. 4

PRKCSH promotes phosphorylation and oligomerization of IRE1α under ER stress. a Immunoblot analysis of IRE1α phosphorylation in L02-Mock and L02-PRK cells treated with 10 μg/mL TM for the indicated time. b, c Immunoblot analysis of IRE1α phosphorylation in L02-WT and L02-PRK KO cells treated with 10 μg/mL TM (b) or glucose-free medium (c) for the indicated time. d, e Immunoblot analysis of IRE1α phosphorylation in PRKCSH-silenced HepG2 (d) and Huh-7 (e) hepatoma cells treated with 10 μg/mL TM for the indicated time. f Immunoblot analysis of IRE1α phosphorylation in Huh-WT and Huh-PRK KO cells treated with 10 μg/mL TM for the indicated time. Phosphorylated IRE1α levels shown below the blots were normalized to total IRE1α. g IRE1α foci in L02-Mock and L02-PRK cells transfected with IRE1α-FV construct and treated with 10 μg/mL TM for 4 h. The percentage of cells with IRE1α foci per total cells with fluorescent IRE1α is shown below the images. The number of IRE1α foci per cell was counted for at least 100 cells with fluorescent IREα-FV. Scale bars represent 5 µm. h IRE1α foci in PRKCSH-silenced Huh-7 cells transfected with IRE1α-FV construct and treated with 10 μg/mL TM for 4 h. PRKCSH is counterstained and its knockdown cells are indicated as dotted line. N indicates the nucleus. Scale bars represent 5 µm. The percentage of cells with IRE1α foci per total cells with fluorescent IRE1α is shown on the right. The number of IRE1α foci per cell was counted for at least 100 cells with fluorescent IREα-FV. i Quantitative real-time PCR analysis of total XBP1 and spliced XBP1 expression in IRE1α-knockdown L02-PRK cells andIRE1α-overexpressing L02-PRK KO cells. j Immunoblot analysis of sXBP1 protein levels in these cells. k Immunoblot analysis of ERK phosphorylation. The numbers indicate the relative levels of sXBP1 and phosphorylated ERKs normalized to the levels of GAPDH or total ERKs, respectively. Quantitative real-time PCR data are shown as mean ± SEM of four independent experiments. One-way ANOVA; **P < 0.01