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. 2019 Dec 19;23(1):100790. doi: 10.1016/j.isci.2019.100790

Figure 5.

Figure 5

USP14 Differentially Regulates IRE1α upon Proteasomal Inhibition

(A) SH-SY5Y cells overexpressing Flag-WT-USP14 or Flag-W58A-USP14 and treated for 6 h with MG132 (20 μM) or tunicamycin (2.5 μg/mL) were subjected to IP with anti-Flag antibodies. Immunoprecipitated complexes were analyzed for the presence of IRE1α and Flag.

USP14 shRNA and scramble shRNA lentivirus-infected SH-SY5Y cells were treated with (B) MG132 (20 μM) or (C) tunicamycin (2.5 μg/mL) for 6 h. Treatment with DMSO served as negative control. The lysates were analyzed for the presence of IRE1α, p-IRE1α, and XBP1s (detected by XBP1 antibody). Immunodetection with anti-USP14 and anti-actin served as a control for silencing efficiency and loading control, respectively. (B) Lower panel depicts densitometry histograms for the ratio of p-IRE1α normalized to total IRE1α and XBP1s normalized to actin. (C) Densitometry ratio of p-IRE1α normalized to total IRE1α.

(B and C) n = 3, p value was calculated by two-way ANOVA. *p ≤ 0.05; **p ≤ 0.01. IRE1α, inositol-requiring protein 1α; XBP1s, spliced X-box-binding protein 1; pIRE1α, phosphorylated IRE1α.