Ab155800

Cells were rinsed three times with prechilled PBS, and then lysed with protein extraction lysate (100 μl/50 ml culture flask) and placed on ice for 30 min. After protein centrifugation at 12,000 rpm for 10 min, the obtained supernatant was subpacked in 0.5 ml centrifuge tubes and store at −20°C or bicinchoninic acid kit for protein quantification. The extracted protein was supplemented with the 6X sodium dodecyl sulfate loading buffer, followed by 10% polyacrylamide gel electrophoresis protein separation. Electroblotted onto a polyvinylidene fluoride membrane, the protein was blocked with 5% skim milk powder, probed with primary antibody against PDIA4 (1:1000; ab155800), β‐actin (1:5000; ab8277; both from Abcam), PI3K/AKT pathway‐related protein PI3K (1:1000; 4255S), phosphorylated (p)‐PI3K (1:1000; 17366S), AKT (1:1000; 4691S) and p‐AKT (1:1000; 13038S; all from Cell Signaling Technology), and next, reprobed with the secondary antibody, goat anti‐rabbit IgG (1:5000; Beijing ComWin Biotech Co., Ltd.). After developing, the expression levels of these proteins were detected. With β‐actin as an internal reference, ImageJ was utilized to calculate the gray value of each protein band.

The cells were lysed with lysis buffer, and the total protein concentration was determined using a bicinchoninic acid protein detection kit (23227, Thermo Fisher Scientific). The protein samples were then diluted with 5 × sample buffer. For detection of protein in the serum samples, serum protein concentration was measured and diluted to 5 μg/uL. Then, the appropriate volume of loading buffer was added, and the mixture was boiled at 100°C for 5 min. Albumin/IgG was removed using the Albumin/IgG Depletion Kit (37591; Qiagen, Germany) according to the manufacturer's instructions. Proteins were separated for 90 min in a 12% separation gel and incubated for 1 h in a PBS blocking solution containing 5% (w/v) evaporated skimmed milk. Next, the cells were incubated with the primary antibodies against DDX17 (1 : 300, PA5-84585, Invitrogen), PDIA4 (1 : 1000, AB155800, Abcam, Cambridge, UK), and β-actin (1 : 5000, AB8277, Abcam) at 4°C, overnight. The membrane was then rinsed and incubated for 1 h with the secondary antibody (1 : 5000, ab114610, Abcam). Finally, proteins were detected using an enhanced chemiluminescence method, and images were captured using a Bio-Spectrum Gel Imaging System (UVP, Upland, CA, USA).

Cardiomyocytes were stained with MitoTracker Deep Red FM (500 nM; M22426, Invitrogen) and fixed in 4% paraformaldehyde (P0099, Beyotime Institute of Biotechnology) at room temperature for 10 minutes. They were then permeabilized with 0.1% Triton 100-X (P0096, Beyotime Institute of Biotechnology) at room temperature for 30 minutes. Cells were washed with phosphate-buffered saline (PBS) three times and blocked with blocking buffer (P0102, Beyotime Institute of Biotechnology) for immunostaining at 37°C for 30 minutes. Samples were incubated with anti-MAP1LC3B antibody (1:100) or anti-ERP72 antibody (ab155800, Abcam; 1:100) at 4°C overnight and then washed in PBS twice before staining with the secondary antibody (31561, Invitrogen; 1:500) at 37°C for 2 hours. Co-localization of fluorescence was measured at 100–400 Hz under the LSCM. Samples without primary antibodies were used as negative controls. Images were analyzed using LAS X software (Leica) and Image-Pro Plus 5.0 (Media Cybernetics). Co-localization represented in Pearson’s correlation coefficient was measured using automatic thresholding, as previously described.⁵⁵ (link)