Anti glyceraldehyde phosphate dehydrogenase gapdh antibody

Marc-145 cells or PAMs were seeded in six-well plates and harvested in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime), and an immunoblotting analysis was performed as previously described [29 (link)]. The cell lysates were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then electroblotted onto pre-equilibrated polyvinylidene difluoride membranes (Millipore). After the membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris [pH 7.5], 150 mM NaCl, 0.5% Tween 20) for 2 h at 37°C, they were rinsed and incubated with an anti-PRRSV N protein monoclonal antibody (SDOW17), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology), horseradish-peroxidase-conjugated anti-mouse IgG antibody (Santa Cruz), and anti-rabbit IgG antibody (Cell Signaling Technology). The signals were detected using ECL reagent (Pierce, Rockford, USA).

Six-well-plate cell samples (2 × 10⁶ cells/well) were harvested in cell lysis buffer (Beyotime, Shanghai, China) containing PMSF (Beyotime, Shanghai, China). Processed samples were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinyl difluoride (PVDF) membrane (Millipore, Boston, MA, USA). Then the membranes were blocked with 5% BSA (Ruishu, China) in TBST (20 Mm Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at 37 °C. After blocking, they were incubated with an anti-PRRSV N protein monoclonal antibody (1:1000 dilution, Jeno Biotech, Inc., Chuncheon-si, South Korea) and an anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing three times with TBST, membranes were incubated with an anti-mouse IgG, HRP-linked antibody or anti-rabbit IgG, HRP-linked antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA) for 1 h at 37 °C. The antibody signals were exposed using an enhanced chemiluminescence (ECL) reagent (Fdbio Science, Guangzhou, China).

Sodium acetate (CAS127-09-3), sodium propionate (CAS137-40-6), sodium butyrate (CAS156-54-7), and disodium succinate (CAS150-90-3) were purchased from Wako (Tokyo, Japan). Recombinant human TNF-alpha (210-TA), IFN-γ (285-IF), and recombinant human IL-13 (213-ILB) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Antibodies against claudin-1 (51-9000), claudin-2 (32-5600), claudin-3 (34-1700), claudin-4 (32-9400), occludin (33-1500), ZO-1 (61-7300 and 33-9100), Alexa 488-conjugated anti-mouse IgG antibody (A11001), and 4′,6-diamidino-2-phenylindole (D3571) were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mounting solution (S3023) was from Dako North America, Inc. (Carpinteria, CA, USA).