Figure 1. Identification of a complex between Ire1α and the Sec61 translocon.
(A) The detergent extracts of either microsomes derived from HEK 293 cells (control) or cells expressing hemagglutinin (HA)-tagged Ire1α were bound to anti-HA resin and eluted with a low pH glycine buffer. The eluted proteins were analyzed by SDS-PAGE and stained with coommassie blue. (B) The cell lysates from non-transfected HEK 293 cells treated with or without DTT were immunoprecipitated (IP) with anti-GFP antibodies as a control or anti-Sec61β antibodies. The bound material was eluted with sample buffer and analyzed along with starting lysates (input, 2% loading) by immunoblotting (IB) using antibodies against the indicated antigens. Calnexin, an abundant endoplasmic reticulum (ER) trans membrane protein was probed as a control. (C) Cell extracts from HEK 293 cells transfected with the indicated FLAG tagged constructs were subject to IP with FLAG antibody. The resulting samples were analyzed by IB with indicated antibodies. (D) HEK 293 cells stably expressing HA-tagged Ire1α were either treated with 10 mM DTT or left untreated for 2 hr. Cells were then semipermeabilized with 0.015% digitonin and treated with the indicated concentration of DSP crosslinker for 30 min at room temperature. Samples were denatured and IP with anti-HA antibodies. The resulting IP was analyzed by IB. Control denotes non-transfected HEK293 cells.
Figure 1—figure supplement 1. Peptides of Sec61α, Sec61β, and Sec61γ identified by mass spectrometry sequences of Sec61α, Sec61β, and Sec61γ annotated to indicate the peptides (yellow) identified by mass spectrometry.
Figure 1—figure supplement 2. Ire1α is codepleted with the Sec61α translocon.
