Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody

Primary HPMECs were obtained from ScienCell (San Diego, CA, USA) and cultured in endothelial cell medium using the recommended supplements from the supplier, and they were used in passages 3 to 5. The Madin-Darby canine kidney cell line was purchased from the American Type Culture Collection (ATCC, VA, USA). The Madin-Darby canine kidneys were cultured in Dulbecco Modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO₂.
Anti-S1PR1 antibody and fluorescein isothiocyanate-conjugated anti-influenza A virus nucleoprotein (NP) antibody were obtained from Abcam (Cambridge, UK). Antibodies against the p65 subunit of NF-κB and phospho-p65 were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Bioworld (St. Louis Park, MN, USA).

Harvested parts of rat seminal vesicles were lysed in triple-detergent radioimmunoprecipitation assay (RIPA) buffer with a protease inhibitor cocktail (Sigma-Aldrich) for 20 min. Total protein was obtained from the supernatant of the lysate after centrifugation at 14 489 g for 10 min at 4°C. The protein quantity was determined by the bicinchoninic acid (BCA) method (Beyotime, Shanghai, China). Quantified protein samples were mixed with sodium dodecyl sulfate (SDS)-polyacrylamide gel-loading buffer (Biyuntian, Shanghai, China) and denatured at 95°C for 5 min. A protein sample (30 μg) was loaded in a lane of a 10% discontinuous SDS-polyacrylamide gel for electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MO, USA). The membranes were blocked with 5% skim milk (BD Biosciences, San Jose, CA, USA) in Tris-buffered saline Tween-20 (TBST) buffer (Biyuntian) for 30 min at ambient temperature and then incubated with anti-tumor necrosis factor-alpha (TNF-α) antibody (1:1000; Abcam, San Francisco, CA, USA), anti-cyclooxygenase-2 (COX-2) antibody (1:1000; Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000; BioWorld, Nanjing, China) overnight. Following incubation with a secondary antibody at ambient temperature for 1 h, signals were visualized by enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA).