Western lightning plus ecl enhanced chemiluminescence substrate kit

Western blotting was conducted as previously described [31 (link)]. The anti-phospho-p38 (Thr180/Tyr182) monoclonal antibody #9211 (Cell Signaling Technology, Danvers, MA, USA) and the rabbit polyclonal anti-β-actin antibody (GeneTex, Irvine, CA, USA) were used to detect Hog1 phosphorylation and Act1, respectively. The horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (GeneTex) was used as the secondary antibody. The blots were visualized using a Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate kit (PerkinElmer) and an ImageQuant LAS 4000 Biomolecular Imager (GE Healthcare Life Science, Marlborough, PA, USA).

The following antibodies were used: Atg7 (Cell Signaling, #2631), p62 (Cell Signaling, #5114), LC3 (Cell Signaling, #4108), CREB (Cell Signaling, #9197), p-CREB (Cell Signaling, #9198), mTOR (Cell Signaling, #2972), p-mTOR (Cell Signaling, #2971), IRS-1 (Cell Signaling, #2382), p-IRS-1 (Cell Signaling, #2381), Akt (Cell Signaling, #4685), p-Akt (Cell Signaling, #4060), p-PERK (Cell Signaling, #3179), PERK (Cell Signaling, #5683), cleaved-ATF-6 (Santa Cruz Biotechnology, #sc-166,659), GAPDH (Santa Cruz Biotechnology, #sc-47,724) and peroxidase goat anti-rabbit IgG (Santa Cruz Biotechnology, #sc-2768). Insulin were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Mouse recombinant CaMKIV were obtained from Sino Biological (Sino Biological Inc. Wayne, PA, USA). Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate Kit (PERKinElmer Inc., Richmond, CA, USA) was used to detect protein expression. Individual protein bands were quantified by ImageJ software. All other chemicals were obtained from standard resource and were of the highest grade available.