Pmsf complete

Soluble whole-cell lysates of myoblasts were prepared using lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer supplemented with 0.2 nmol/L PMSF (Complete, Roche, Shanghai, China). The lysates were denatured at 95 °C for 5 min and 10 mg of protein samples were subjected to 10% polyacrylamide gel. Blots were transferred to PVDF membranes and incubated in the primary antibodies, including DESMIN (1:50, DSHB, Iowa City, IA, USA), mouse, TET2 (1:400, Abcam, Fremont, CA, USA), MyHC (1:50, DSHB, Iowa City, IA, USA), α-TUBLIN (1:5000, Novus Biologicals Littleton, CO, USA). HRP-conjugated goat anti-rabbit and anti-mouse IgG antibodies (1:5000, Cell Signaling, Beverly, MA, USA) were used as secondary antibodies, respectively. Then, blots were detected using ECL reagents (Thermo Fisher Scientifc, Waltham, MA, USA). The quantity of detected proteins was normalized to that of α-TUBLIN using ImageJ 1.8 software (National Institutes of Health, Bethesda, MD, USA).

Freshly isolated brains were homogenized in lysis buffer (150mM NaCl, 50mM Hepes pH7.6, 1% NP-40, 0,5% sodium deoxycholate, 5mM EDTA) supplemented with 1mM PMSF, Complete (Roche), 20mM NaF, 10mM b-glycerophosphate, 5mM Na-pyrophosphate, and 1mM Na-orthovanadate. Equal amounts of protein were separated by electrophoresis (Novex NuPAGE Bis-Tris protein gel 4–12% or 5% home-made Bis-Tris gel) and transferred to a PVDF membrane in 20% methanol transfer buffer. Membranes were probed with polyclonal rabbit antibodies to PW1 (rabbit, 1:10,000) (Relaix et al., 1996) and GAPDH (Abcam). Antibody binding was visualized using horse-radish peroxidase (HRP)-conjugated species-specific secondary antibodies (Jackson ImmunoResearch) followed by enhanced chemiluminescence (Pierce).