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. 2020 Jan 27;219(2):e201904048. doi: 10.1083/jcb.201904048

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© 2020 Grey et al.

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Figure 3. — Suppression of IRE1α expression does not explain how IRE1β affects the UPR. (A–C) IRE1α expression was assayed by (A) qPCR for mRNA or (B) immunoblot for protein in HEK293^doxIRE1β cells treated with Tg for indicated time points (immunoblots are representative of two or three independent experiments). The anti-βactin blot for the 3-h Tg treatment (fourth row down) is duplicated from Fig. 1 C, as the same experiment and membrane was used to probe for anti-FLAG (Fig. 1 C), anti-IRE1α, and anti-βactin. The actin normalized band intensities are plotted in C. For A and C, data are plotted as log2 fold change relative to 0 ng/ml Dox without Tg. Each experiment for a given time point included a control sample without Tg. (D) Top: Schematic of human IRE1α gene (ERN1) promoter region and luciferase reporter constructs. Putative XBP1 binding sites are indicated as boxes. (Bottom panel) IRE1α-Luc reporter activity in HEK293T cells (left) cotransfected with either control vector or XBP1 expression vector or (right) treated with SubA_A272B or SubAB (100 ng/ml) for 8 h (n = 3). (E) IRE1α protein and mRNA levels in HEK293^doxIRE1β cells transfected with IRE1α siRNA or control siRNA and treated with Tg (n = 2). (F) Same as in E for spliced XBP1 mRNA. Bars and error bars represent mean values ± SEM; significance is indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).