Mouse anti beta actin

Protein was extracted from differentiated myoblasts using NCH buffer (4% sodium dodecyl sulphate, 4 M urea, 150 mM Tris) diluted 1:1 with RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma) containing a complete protease inhibitor cocktail (1:100, Roche). Samples were collected and boiled for 3 minutes and 30 μl of each sample was run on a NuPAGE Novex 3–8% Tris-Acetate Gel, with constant voltage of 150 V for 1 hour, before transfer to a nitrocellulose membrane at 300 mA for 2 hours. The membrane was blocked in Odyssey blocking solution (LI-COR Biosciences) for 60 min, before incubation with rabbit anti-dystrophin (1:2000, Fisher Scientific) or rabbit anti-FLAG-tag (1:2000, Sigma) and mouse anti-beta-actin (1:4000, Invitrogen) overnight at 4 °C. The membrane was washed with PBS containing 1% Tween 20 (PBST) before incubation with IRDye 680 RD goat anti-rabbit and IRDye 800CW goat anti-mouse 2nd antibodies (1:15000, LI-COR Biosciences) for 1 hour at room temperature. The fluorescent image was acquired by Odyssey Clx infrared imaging system (LI-COR Biosciences) using image studio software 3.1.4.

Cells were lysed with 10 mmol/L Tris at pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% SDS, and protease/phosphatase inhibitor cocktail (#78440, Thermo Fisher). Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, blotted with primary antibodies overnight at 4 °C, and detected using horseradish peroxidase-conjugated secondary antibodies followed by exposure to chemiluminescence reagents (#PI34580, Thermo Fisher). The following antibodies were used: rabbit anti-HIF1α (#10006421, 1:500, Cayman), goat anti-HIF2α (#AF2997, 1 µg/ml, R&D Systems), mouse anti-beta actin (#MA1-91399, 1:50,000, Invitrogen), HRP-linked anti-rabbit IgG (#7074, 1:10,000, Cell Signaling), HRP-linked anti-mouse IgG (#7076, 1:60,000, Cell Signaling), and HRP-linked anti-goat IgG (#705035147, 1:20,000, Jackson ImmunoResearch).

Protein was extracted from differentiated myoblasts using NCH buffer (4% sodium dodecyl sulphate, 4M urea, 150 mM Tris) diluted 1:1 with RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma) containing a complete protease inhibitor cocktail (1:100, Roche). Samples were collected and boiled for 3 minutes and 30 μl of each sample was run on a NuPAGE Novex 3–8% Tris-Acetate Gel, with constant voltage of 150 V for 1 hour, before transfer to a nitrocellulose membrane at 300 mA for 2 hours. The membrane was blocked in Odyssey blocking solution (LI-COR Biosciences) for 60 min, before incubation with rabbit anti-dystrophin (1:2000, Fisher Scientific) or rabbit anti-FLAG-tag (1:2000, Sigma) and mouse anti-beta-actin (1:4000, Invitrogen) overnight at 4 °C. The membrane was washed with PBS containing 1% Tween 20 (PBST) before incubation with IRDye 680 RD goat anti-rabbit and IRDye 800CW goat anti-mouse 2nd antibodies (1:15000, LI-COR Biosciences) for 1 hour at room temperature. The fluorescent image was acquired by Odyssey Clx infrared imaging system (LI-COR Biosciences) using image studio software 3.1.4.