Xcell surelock mini cell modules

For protein analysis, cells were lysed in M-PER buffer (Thermo Scientific) plus complete EDTA-free protease inhibitor (Roche) and phosphatase inhibitor cocktail (Sigma). Protein concentration was determined using BCA Protein Assay (Thermo). Western blots were performed using 10% and 12% Bis-Tris precast gels (NuPage) on Invitrogen XCell SureLock® Mini-Cell modules. Gels were run using MES buffer and transferred onto nitrocellulose transfer membrane using an XCell II™ Blot Module. Antibody binding was detected by using near-infrared-dye-conjugated secondary antibodies (Licor) on the LI-COR Odyssey scanner or visualized by capturing on a CL-XPosure film using ECL SuperSignal West Dura Extended Duration Substrate (both from Thermo Scientific). See Supplemental Experimental Methods for details of the antibodies used.

For protein analysis, cells were lysed in T-PER buffer (Thermo Fisher Scientific 78510) plus phosphatase inhibitor cocktail (Cell Signaling Technology #5872S). Protein concentration was determined using Pierce BCA Protein Assay (Thermo Fisher Scientific 23225). Western blots were performed using 10% and 4–12% gradient Bis-Tris precast gels (NuPage) on Invitrogen XCell SureLock Mini-Cell modules. Gels were run using 2-(N-morpholino)ethanesulfonic acid (MES) buffer (Invitrogen NP000202) and transferred onto nitrocellulose transfer membrane using an XCell II Blot Module or iBlot Dry Blotting System (Thermo Fisher Scientific). Antibody binding was visualized on CL-XPosure film using ECL SuperSignal West Extended Duration Substrate (both from Thermo Fisher Scientific) or using the Odyssey CLx Imaging System (LI-COR Biosciences). Antibodies used: ATF4 (Cell Signaling Technology #11815), BiP (CST #3177), CHOP (CST #2895), eIF2a (CST #9722), phospho-eIF2a (CST #3398), IRE1α (CST #3294), PERK (CST #3192), PERK p-T980 (CST #3179), Spliced XBP-1 (BioLegend #619502), actin (Sigma A5441 1:3000).