Fig. 5

The dimerization and stability of IRE1α regulated by TMCO1 depends on the overload of ER Ca²⁺ store. a qRT-PCR analysis of IRE1α mRNA in WT or TMCO1 KD cells treated with/without 2.5 μM ACTD for 5 h. Bar graphs represent the mean ± SEM from three independent assays. **P < 0.01, ***P < 0.001. b Western blotting analysis of IRE1α levels in WT and TMCO1 KD cells treated with/without 2.5 μM ACTD for 5 h. GAPDH was used as a loading control. Relative quantification of IRE1α levels was shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01. c The stability of IRE1α was measured after the treatment of 100 μg/ml CHX for the indicated times, followed by western blotting analysis. GAPDH was used as a loading control. Relative quantification of IRE1α levels was shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. d The stability of IRE1α was measured after the treatment of 100 μg/ml CHX along with/without 20 μM KIRA6 for the indicated times, followed by western blotting analysis. GAPDH was used as a loading control. Relative quantification of IRE1α levels was shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. *P < 0.05, n.s., no significance. e The digitonin lysate of WT, TMCO1 KD cells, TMCO1 KD cells transfected with TMCO1 or TMCO1-D140A-IRES-mCherry were analyzed by BN-PAGE immunoblotting with IRE1α antibody. GAPDH was used as a loading control. f The stability of IRE1α in WT, TMCO1 KD cells. TMCO1 KD cells transfected with TMCO1 or TMCO1-D140A-IRES-mCherry were treated with 100 μg/ml CHX for the indicated times, followed by immunoblotting. GAPDH was used as a loading control