Horseradish peroxidase conjugated goat anti mouse or anti rabbit igg fab

The cells were lysed in lysis buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, a protease and phosphatase inhibitor mixture (Roche)] and centrifuged for 15 min 20,000×g at 4 °C to obtain the cell lysates. Next, the cell lysate concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). The total protein sample was prepared using 5× sodium dodecyl sulfate (SDS) sample buffer and heated at 99 °C for 10 min. Proteins were separated on a 10% SDS–polyacrylamide electrophoresis gel and transferred to a 0.45-μm pore size nitrocellulose membrane (GE Healthcare Life Science). The membrane was incubated overnight at 4 °C with primary antibodies in TBS-T [150 mM NaCl, 20 mM Tris–HCl (pH 8.0), and 0.05% Tween-20] containing 3% BSA, followed by secondary antibody incubation using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Fab) (Enzo Life Sciences) in 5% skim milk dissolved in TBS-T at 26 °C for 2 h. Proteins were visualized with an ECL western blotting reagent (BioNote) and analyzed on a Fusion Solo-S image analyzer (Vilber). Protein band intensities were quantified and analyzed using ImageJ software.

The primary antibodies used for immunoblotting and immunofluorescence were as follows: rabbit polyclonal anti-IK (Bethyl Laboratories, A301-708A), mouse monoclonal anti-β-actin (Santa Cruz, sc-47778), mouse monoclonal anti-Pax-7 (Santa Cruz, sc-81648), mouse monoclonal anti-MyoD (Santa Cruz, sc-377460), mouse monoclonal anti-myogenin (Santa Cruz, sc-52903), rabbit polyclonal anti-MyHC (Santa Cruz, sc-20641), mouse polyclonal anti-PARP (Cell Signaling, #9542), rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) (Cell Signaling, #9661), rabbit monoclonal anti-Cleaved Caspase-9 (Asp315) (Cell Signaling, #20750), anti-GFP (Cell Signaling, #2956), anti-Bungarotoxin Alexa Fluor™ 647 conjugate (Invitrogen™, #B35450), anti-fast-twitch muscle (DSHB, F310), and anti-slow-twitch muscle (DSHB, F59). The horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Fab) secondary antibodies were purchased from Enzo Life Sciences.