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. 2022 Nov 5;23(21):13586. doi: 10.3390/ijms232113586

Figure 1.

Figure 1

Gsk-3 negatively regulates ATF4 protein expression in mouse β cells under ER stress. (a) Isolated mouse islets were incubated with tunicamycin (5 μg/mL) for 14 h and Akt-Gsk-3 signalling was analysed by Western blot. Relative pGsk-3β/Gsk-3β are shown in the graph as mean ± SD (n = 3). *** p < 0.001 by unpaired student’s t-test. (b) Isolated islets were incubated with tunicamycin for 14 h in the presence or absence of GSK-3 inhibitor, SB216763 (5 μM) and protein lysates were analysed by Western blotting using indicated antibodies. Relative expressions of ATF4 are shown as mean ± SD (n = 3). ** p < 0.01 by one-way ANOVA followed by Bonferroni’s post hoc test. MIN6 in which Gsk-3 activity was inhibited by (c) SB216763 (5 μM) or (d) by retrovirus-mediated induction of HA-Gsk-3βKM were incubated with tunicamycin (5 μg/mL) for indicated periods. Protein lysates were analysed by Western blotting. Relative expression of ATF4 to the basal are expressed as mean ± SD (n = 3, respectively). ** p < 0.01, *** p < 0.001 by two-way ANOVA with Bonferroni’s post hoc test. (e) The nuclear extracts of MIN6 stably expressing GFP or HA-Gsk-3βKM were prepared after incubation with tunicamycin at indicated time points and were analysed by Western blotting using indicated antibodies. Representative images of two independent experiments are shown.