Chemidoc xrs western blot imaging system

For protein extraction, mouse TA muscles were disrupted by Tissue Lyser LT (Qiagen, Hilden, Germany) in RIPA buffer (140 mM NaCl, 3 mM MgCl₂, 1 mM EDTA, and 15 mM HEPES [pH 7.2], also containing 0.5% sodium deoxycholate, 1% NP-40, and 0.1% SDS) supplemented with a cocktail of protease inhibitors (Roche, Sigma-Aldrich) and phosphatase inhibitors. Cultured cells were lysed on plates with the same RIPA buffer. Western blots were carried out using the following antibodies: mouse mAb to MYOG (F5D), mouse mAb to TetR (Clone 9G9) from Takara Bio (Kusatsu, Japan), and mouse mAb to Cas9 (7A9-3A3) and rabbit polyclonal antibody to p38α (C-20) from Santa Cruz Biotechnology (Santa Cruz, CA). After incubation with primary antibodies, filters were incubated with horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies from Santa Cruz Biotechnology and revealed with a chemiluminescence detection system by Cyanagen (Bologna, Italy). Imaging was carried out by the ChemiDoc XRS Western Blot Imaging System using ImageLab 4.0 software (Bio-Rad).

Cells were lysed in RIPA buffer (140 mM NaCl, 3 mM MgCl2, 1 mM EDTA, and 15 mM HEPES, pH 7.2, also containing 0.5% sodium deoxycholate, 1% NP-40, and 0.1% SDS) supplemented with a cocktail of protease inhibitors (Roche, Sigma-Aldrich, St. Louis, MO). Western blots were carried out using horseradish-peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies and revealed with a chemiluminescence detection system by Cyanagen (Bologna, Italy). Imaging and quantitation of the bands were carried out by the ChemiDoc XRS Western Blot Imaging System using the ImageLab 4.0 software (Bio-Rad, Hercules, CA).