Anti pperk

The following antibodies were purchased from Cell Signaling Technology: anti-IRS-1 (2382), anti-pAKT S473 (9271), anti-p85 (4292), anti-IGF-1Rβ (9750), anti-PDK1 (13037). Other antibodies were from the following commercial sources: anti-VAPB (Sigma-Aldrich, HPA013144), anti-NOGOA (Bio-Rad, AHP1799), anti-BAP31 (Santa Cruz Biotechnology, sc-48766), anti-PERK (abcam, ab229912), anti-pPERK (Affinity Biosciences, DF7576), anti-AKT (HUABIO, ET1609-47), anti-Actin (HUABIO, M1210-2), anti-Tubulin (HUABIO, M1305-2), anti-GFP (HUABIO, ET1607-31), anti-FLAG (YEASEN, 30503ES60), anti-HA (Invitrogen, PA1-985) and anti-mCherry (ABclonal, AE002).

Total protein was extracted from cells with RIPA lysis buffer (Beyotime, China) after incubation for 48 h. After quantification of the protein concentration with a BCA protein assay kit (Beyotime, China), proteins in the samples (40 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% fat-free milk and incubated with diluted primary rabbit polyclonal antibodies, including anti-GHSR-1a (1:1000, Affinity, USA), anti-PERK (1:1000, Affinity, USA), anti-p-PERK (1:2000, Affinity, USA), anti-AFT4 (1:1000, Affinity, USA), anti-CHOP (1:1000, Affinity, USA), and anti-GAPDH (1:1000, Hangzhou Xianzhi Biology Co., Ltd., China) antibodies, overnight at 4 °C. Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Boster, China) as the secondary antibody for 2 h at room temperature. The bands were visualized by ECL and quantified using Bandscan software 5.0 (Glyko Inc., CA, USA).