Quantitative Western Blot Analysis of ER Stress Signaling

Preparation of cell and tissue lysates and Western blot were performed as previously described^{27 (link)}. IRE1α phosphorylation was measured by a Phos-tag-based Western blot method^{27 (link)–29 (link)}. For non-reducing SDS-PAGE, lysates were prepared in 5 X non-denaturing sample buffer (250 mM Tris HCl pH 6.8, 1% SDS, 50% glycerol and 0.05% bromophenyl blue) without boiling prior to be separated on a SDS-PAGE gel. Antibodies used in this study were: HSP90 (sc-7947, 1:6,000), GFP (sc-8334, 1:2000), BiP (sc-1051, 1:1,000), IRE1β (sc-20575, 1:1000), JNK1 (sc-571, 1:1000), α-Tubulin (sc-5286, 1:2000) from Santa Cruz; IRE1α (#3294, 1:2000), PERK (#3192, 1:2,000), Caspase-3 (#9665, 1:1000), p-T183/Y185-JNK (#9255, 1:2000), from Cell Signaling; Sel1L (ab78298, 1:2,000), OS9 (ab109510, 1:10,000) from Abcam; Hrd1 (NB100-2526, 1:8,000) from Novus Biologicals; Calnexin (SPA-860, 1:8,000) and PDIA1 (SPA-890, 1:8,000) from Enzo Life Sciences; Flag-HRP (A-8592, 1:8,000), HA (H9658, 1:5,000) from Sigma. Antibodies for Bag6 (rabbit, 1:10,000) and H2A (rabbit, 1:10,000) were kind gifts from Dr. Yihong Ye (NIDDK). Band density was quantitated using the Image Lab software on the ChemiDOC XRS⁺ system (Bio-Rad). Protein levels were normalized to HSP90 or α-Tubulin and are presented as mean ± s.e.m. unless otherwise specified.