Anti tubulin

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).

The protein was extracted from heart tissues using RIPA lysis buffer; Then, the protein concentration was measured by the BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Afterwards, the protein was separated on 10% SDS-PAGE, and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA), membranes were blocked with 5% skim milk powder (cat. no.BS102; Biosharp, Wuhan, China) for 2 h and then incubated with the anti-Mapk14 (1:1000, Affinity Biosciences, cat. no. AF6456, Jiangsu, China), anti-Hif1a (1:1000, cat. no. #48085; Cell Signaling Technology, MA, USA), anti-Myc (1:500, cat. no. A1309; ABclonal, Wuhan, China), anti-Hsp90ab1 (1:2000, cat. no. ET1605-56; Huabio, Hangzhou, China), anti-Hsp90aa1 (1:1000, cat. no. ET1605-57; Huabio, Hangzhou, China) and anti-Tubulin (1:1000, cat. no. AC030; ABclonal, Wuhan, China) primary antibodies at 4 °C overnight. Subsequently, membranes were washed with TBST and incubated with the HRP-labeled secondary antibody (1:3000; cat.no. S0001; Affinity Biosciences, Jiangsu, China) for 2 h. The bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was quantified by densitometry using Image J (NIH, Bethesda, MD, USA).