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. Author manuscript; available in PMC: 2021 Jan 15.
Published in final edited form as: Cell Rep. 2020 Dec 29;33(13):108563. doi: 10.1016/j.celrep.2020.108563

Figure 2. Sec63 Inhibits IRE1α Clustering during ER Stress in Cells.

Figure 2.

(A) HEK293 IRE1α−/− cells complemented with IRE1α-HA were transfected with control, Sec63, or Sec62 siRNA. The expression of IRE1α-HA was induced with 5 ng/mL doxycycline. After 30 h of transfection, the cells were either left untreated or treated with 5 μg/mL Tg for 1.5 h and processed for immunostaining with anti-HA antibodies for IRE1α. Scale bars, 10 μm.

(B) Quantification results of the number of cells with IRE1α clusters in (A). Error bars represent the standard error of the mean (SEM) from two independent experiments.

(C) HEK293 IRE1α−/− cells were treated with the indicated siRNAs as in (A) but analyzed by immunoblotting for the indicated antigens.

(D) HEK293 IRE1α−/− cells expressing IRE1α-HA were transfected with empty vector, WT Sec63, or the J-domain mutant of Sec63. The expression of IRE1α-HA was induced with 5 ng/mL doxycycline. After 24 h of transfection, the cells were either left untreated or treated with 5 μg/mL Tg for 1.5 h and subsequently processed for immunostaining with anti-HA antibodies for IRE1α.

(E) Quantification results of the number of cells with IRE1α clusters in (D). Error bars represent the SEM from two independent experiments.

(F) Cells were transfected as in (D) but analyzed by immunoblotting for the indicated antigens.

(G) HEK293 IRE1α−/− cells expressing either IRE1α-HA or IRE1α-CNX-TMD-HA were induced with 5 ng/mL doxycycline and treated with either Tg or Tm for the indicated time points. The cells were then processed for immunostaining as in (A).

(H) Quantification results of the number of cells with IRE1α clusters in (G). Error bars represent the SEM from two independent experiments.

See also Figure S2.