Fig. 4

ER Ca²⁺ overload reduces the interaction between IRE1α and BiP. a The levels of XBP1s in HEK293T-IRE1α knockout cells transfected with IRE1α or IRE1α deleted LD were measured after treating with 1 μM TG for 2 h, followed by western blotting analysis. GAPDH was used as a loading control. Relative quantification of XBP1s levels was shown in the right panel. Data are shown as the mean protein intensity normalized to GAPDH ± SEM from 3 independent experiments. ***P < 0.001. EV, empty vector. ∆LD, deleted LD of IRE1α. b Expression levels of calnexin were analyzed by western blotting in the wild-type or TMCO1 KD cells. GAPDH was used as a loading control. c Pull down assay assessing the effects upon addition of increasing concentrations of Ca²⁺ to GST-tagged BiP-NBD and FLAG-tagged IRE1α-LD complex. 1.2 mM Ca²⁺ disrupted IRE1α-luminal domain interaction and causes the BiP-ATPase domain to dissociate. Relative quantification of LD-FLAG level was shown in the right panel. Data are shown as mean ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test: ***P < 0.001. d The resting G-CEPIA1er fluorescence ratio signals were detected in HEK293T treated with 1 μM TG or 5 μM CDN1136 for 2 h. Ctrl, n = 103. TG, n = 171. CDN1136, n = 160. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001. e The resting G-CEPIA1er fluorescence ratio signals were detected in HEK-293 T treated with/without 100 μM 2-APB for 2 h. Ctrl, n = 105. 2-APB, n = 165. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001. (f and g) HEK-293 T cells transfected with BiP-Flag were treated with 1 μM TG or 5 μM CDN1136 (f) or 100 μM 2-APB (g) for 2 h followed by a co-immunoprecipitation for Flag. Protein loading was normalized to FLAG and relative co-immunoprecipitated IRE1α was examined via immunoblotting