Peroxidase goat anti rabbit igg

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.

All chemicals used were of analytical grade and were purchased from Sigma (St. Louis, MO) unless otherwise stated. The following antibodies were used: anti-Atg7, anti-LC3, anti-p-PERK, anti-PERK, anti-p-IR, and anti-IR (Cell Signaling Technology Inc., Danvers, MA); anti-G6Pase, anti-Pck1, anti-p62, anti-p-Akt, anti-Akt, anti-p-eIF2α, anti-eIF2α, anti-GAPDH, and peroxidase goat anti-rabbit IgG and peroxidase goat anti-mouse IgG from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., CA).

Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo) according to the manufacturer’s instructions. Twenty μg of protein were separated on 10% SDS-PAGE gel and electro-transferred onto NC membranes. The membranes were incubated with ABCC4 or GAPDH mouse monoclonal antibodies (Santa Cruz Biotech, USA) and subsequently with a peroxidase goat anti-rabbit IgG (Santa Cruz Biotech). The membranes were then developed with Clarity Western ECL substrates (Merck Millipore) and visualized with the ChemiDocTM MP Imaging System (Bio-Rad).