Horseradish peroxidase conjugated anti mouse igg antibody

MHY3200 was designed and synthesized by Prof. H. R. Moon at the College of Pharmacy, Pusan National University, Busan, Korea [7 (link)], and dissolved in dimethyl sulfoxide (DMSO). Other chemical reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes Inc. (Eugene, OR, USA). ECL Western Blotting Detection Reagents, Pierce bicinchoninic acid (BCA) protein assay kit, polyvinylidene difluoride (PVDF) membrane, and Dokdo-MARK™ protein size marker were purchased from GE Healthcare Biosciences (Buckinghamshire, UK), Thermo Fisher Scientific (Rockford, IL, USA), the Millipore Corporation (Bedford, MA, USA), and ElpisBiotech (Daejeon, Korea), respectively. Anti-NOX4, anti-p-Akt (Ser 473), manganese superoxide dismutase (MnSOD), p-p65 (Ser 276), p65, β-actin, transcription factor IIB (TFIIB), and cyclooxygenase-2 (COX-2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-FoxO1 (Ser 256) were purchased from Cell Signaling Technology (Danvers, MA, USA) while anti-rabbit IgG-horseradish peroxidase-conjugated antibodies, anti-mouse IgG-horseradish peroxidase-conjugated antibodies, and anti-goat IgG-horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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).

Protein lysates of fibroblasts were prepared using the Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). Equal amounts of protein lysates were subjected to sodium dodecyl sulfate‐polyacrylamide electrophoresis with 4%‐20% gradient gels (Bio‐Rad Laboratories) and transferred to polyvinylidene difluoride membranes using the Trans‐Blot Turbo Transfer System (Bio‐Rad Laboratories). After blocking with Block Ace (DS Pharma Biomedical), the membrane was incubated with the following primary antibodies: mouse anti‐human Endo180 antibody (Santa Cruz Biotechnology) or mouse anti‐GAPDH antibody (Abcam) for 2 hours at 25°C. After washing in phosphate‐buffered saline with 0.05% Tween‐20 (PBS‐T), the membrane was incubated with horseradish peroxidase‐conjugated anti‐mouse IgG antibody (Santa Cruz Biotechnology) for 1 hour at 25°C. After washing in PBS‐T, the blots were detected with the ECL‐PRIME (GE Healthcare) and ChemiDoc XRS systems (Bio‐Rad Laboratories). Endo180 production was expressed as a percentage of the level in control cells.

After the chromatographic separation of gangliosides, the plates were soaked in acetone plus 0.1% polyisobutylmethacrylate. After drying and blocking with PBS-4% milk, the plates were incubated with 5 μg/ml of anti-Neu5Gc-GM3 murine 14 F7 antibody [31 (link), 32 (link)], overnight, and, then, with a horseradish peroxidase-conjugated anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The reaction was stained with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Dallas, MA, USA).