SDS-PAGE and Western Blotting Protocol

SDS-PAGE and western blotting was conducted as previously described (25 (link)). Briefly, cell lysates were evaluated to determine protein levels and then loaded on SDS-PAGE gel. The protein bands were transferred to a membrane, which was subsequently incubated with primary antibodies (Abs), including anti-IRE1α Ab (3294S, Cell Signaling Technologies, MA, USA) or anti-PERK Ab (3192S, Cell Signaling Technologies), and then with secondary Abs including HRP-linked anti-rabbit IgG Ab (Cell Signaling Technologies, MA, USA), and HRP conjugated polyclonal goat anti-mouse IgG F(ab′)2 (Enzo Life Sciences, NY, USA). The substrate for enhanced chemiluminescence (ECL) was used from femtoLUCENT™ PLUS HRP Kit (G-biosciences, MO, USA). Images were obtained with ImageQuant™ LAS 4000 mini system (GE Healthcare Life Sciences, Buckinghamshire, UK) and analyzed using the Image J software (NIH, Bethesda, MD, USA).