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. 2017 May 15;6:e27187. doi: 10.7554/eLife.27187

Figure 5. The Sec61 translocon regulates the attenuation of IRE1α activity during ER stress.

(A) IRE1α -/- HEK293 cells complemented with either wild type IRE1α-HA or wIRE1α-HA were treated with 2.5 μg/ml of Tg for the indicated time points and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. (B) Quantification of IRE1α and wIRE1α phosphorylation from panel A. (C) IRE1α-HA or wIRE1α-HA cells were treated with 10 μg/ml of TM for the indicated time points and analyzed as in panel A. (D) Quantification of IRE1α and wIre1 phosphorylation from panel C. (E) IRE1α-HA or wIRE1α-HA cells were transfected with XBP1s plasmid and treated with 1 μg/ml of Tg for the indicated time points and analyzed as in panel A. (F) Quantification of IRE1α and wIRE1α phosphorylation from panel E. (G) IRE1α-HA or sIRE1α-HA cells were treated with 2.5 μg/ml of Tg for the indicated time points and analyzed as in panel A. (H) Quantification of IRE1α and sIRE1α phosphorylation from panel G.

DOI: http://dx.doi.org/10.7554/eLife.27187.015

Figure 5—source data 1. Attenuation of IRE1α and wIRE1α in Tg-treated cells as described in Figure 5B.
DOI: http://dx.doi.org/10.7554/eLife.27187.016
elife-27187-fig5-data1.xlsx (36KB, xlsx)
DOI: 10.7554/eLife.27187.016
Figure 5—source data 2. Attenuation of IRE1α and wIRE1α in TM-treated cells as described Figure 5D.
DOI: http://dx.doi.org/10.7554/eLife.27187.017
elife-27187-fig5-data2.xlsx (31.7KB, xlsx)
DOI: 10.7554/eLife.27187.017
Figure 5—source data 3. Attenuation of IRE1α and wIRE1α in XBP1s expressing cells as described Figure 5F.
DOI: http://dx.doi.org/10.7554/eLife.27187.018
elife-27187-fig5-data3.xlsx (35.3KB, xlsx)
DOI: 10.7554/eLife.27187.018
Figure 5—source data 4. Attenuation of IRE1α and sIRE1α in Tg-treated cells as described in Figure 5H.
DOI: http://dx.doi.org/10.7554/eLife.27187.019
elife-27187-fig5-data4.xlsx (36.3KB, xlsx)
DOI: 10.7554/eLife.27187.019

Figure 5.

Figure 5—figure supplement 1. Attenuation of the endogenous IRE1α activity during ER stress.

Figure 5—figure supplement 1.

(A) HEK293 cells were treated with 5 ug/ml tunicamycin (TM) for the indicated times and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. (B) Quantification of endogenous IRE1α phosphorylation from panel A.
DOI: http://dx.doi.org/10.7554/eLife.27187.020
Figure 5—figure supplement 2. Accumulation of misfolded proteins is required for the activation of IRE1α.

Figure 5—figure supplement 2.

IRE1α -/- HEK293 cells stably expressing IRE1α or wIRE1α were pretreated with 5 μg/ml TM for 3 hr, washed, and chased without TM but including 100 μg/ml cycloheximide (CHX) for the indicated time points and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens.
DOI: http://dx.doi.org/10.7554/eLife.27187.021