Figure 2. IRE1α forms a hetero-oligomeric complex with the Sec61 translocon.
(A) Coomassie blue stained gels showing IRE1α variants that were purified from HEK293 cells stably expressing 2X strep-tagged IRE1α. (B) The indicated concentration of purified IRE1α proteins was analyzed by BN-PAGE based immunoblotting with IRE1α antibodies. (C) The purified IRE1α proteins were analyzed as in panel B using Sec61α antibodies. (D) The purified IRE1α proteins were analyzed by standard immunoblotting with BiP and Sec61α antibodies.
Figure 2—figure supplement 1. XBP1u mRNA cleavage by purified IRE1α variants.
(A) The purified IRE1α proteins were independently incubated with 32P-labeled unspliced XBP1u mRNA prepared from in vitro transcription reactions. The cleaved fragments were separated by 6 M urea PAGE and exposed for autoradiography. (B) Immunoblots of purified Ire1a proteins. Note that both wild type IRE1α and strong Ire1a (sIRE1α) contain the Sec61 translocon, whereas weak Ire1a (wIRE1α) lacks the Sec61 translocon.