Mouse anti flag tag monoclonal antibody

Stable transfected ADSCs were selected with puromycin (Sigma-Aldrich, St. Louis, MO, USA) and CNGRC expression levels of cells at the third passage (12 days after transduction) were identified by western blot analysis. Total protein was extracted from ADSCs transfected with LV-EGFP and LV-CNGRC-3Flag-EGFP, respectively. Following denaturation, the protein samples were centrifuged at 12,000 × g for 2 min. The target proteins were hybridized with mouse anti-flag tag monoclonal antibody (1:1,000; Proteintech, Chicago, IL, USA), mouse anti-β-actin monoclonal antibody (1:5,000; Proteintech) and horseradish-peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (1:10,000; CWBio, Beijing, China) IgG. Enhanced chemiluminescent substrates (Pierce Biotechnology, Inc., Rockford, IL, USA) were used to detect the signals of targeted proteins.

Cells were harvested with trypsin-ethylenediaminetetraacetic acid (0.25%) (Hyclone), lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, and 1% Triton X-100, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with Roche complete protease inhibitor cocktail on ice for 1 h, and then centrifuged (12,000× g, 10 min, 4 °C) to remove cell debris and insoluble materials. The supernatant was collected as the total cellular protein. Protein concentration was measured using a bicinchoninic acid assay (Beyotime Biotechnology, Wuhan, China). Proteins were separated in a 10% denaturing Tris-glycine gel, and Western blot analysis was performed using the corresponding antibodies, including mouse anti-Flag-tag monoclonal antibody (1:5000 dilution, Protein Tech, Wuhan, China). The anti-IRE1α (1:1000 dilution, NB100-2324, Novus, Briarwood, NY, USA), anti-IRE1α-p (1:1000 dilution, NB100-2323, Novus), and anti-GRP78 (1:1000 dilution, NBP1-06274, Novus) were purchased from NOVUS; anti-Caspase 3 (1:1000 dilution, 66470-2-Ig, Proteintech, Wuhan, China) and anti-cleave caspase 3 (1:1000 dilution, MA9132, Abmart) were purchased from Proteintech and Abmart company, respectively. GAPDH (1:5000 dilution, # 2118S, CST, Danvers, MA, USA) was purchased from Cell Signaling Technology. Protein band intensity was quantified using Image J software (Image J (nih.gov), accessed on 15 April 2021).