Sc 53091

Muscles samples were homogenized (Polytron 2100; Lucerne, Switzerland) for 3 × 30 s on ice in Radioimmunoprecipitation assay buffer (R0278-50 ml, Sigma-Aldrich) supplemented with protease inhibitors (1:100; P8340, Sigma-Aldrich Company Ltd. Gillingham, United Kingdom). Following bicinchoninic acid assay quantification (23 227, ThermoFisher Scientific, Waltham, Massachusetts, United States), 30–50–100 μg of total protein were heat denatured for 5 min at 100°C before loading onto NuPAGE 3–8% TRIS Acetate Midi Gel (Novex, Life Technologies) and transferred to Polyvinylidene difluoride membranes (Millipore, Burlington, Massachusetts, United States). Membranes were blocked for 1 h with Odyssey Blocking buffer (926-41090, LI-COR; USA) and then incubated with primary antibodies in Odyssey Blocking buffer PBS + 0.1% Tween for 2 h at room temperature. Primary antibodies used were mouse monoclonal anti-utrophin [1:50, MANCHO3(84A), gift from G.E. Morris], rabbit polyclonal anti-dystrophin (1:200; ab15277, Abcam) and mouse monoclonal anti-MYH3 (1:100; sc-53091, Santa Cruz Biotechnology). The Odyssey Imaging System (LI-COR Biosciences, USA) was used to read infrared fluorescence of the secondary antibodies. Relative expression of the target proteins was quantified using vinculin as references and the Image Studio Lite Ver 5.0 software (LI-COR Biosciences).

After cells attached to the slide, the culture medium was discarded and the cells were washed with PBS buffer three times. The cells were fixed with 4% cold paraformaldehyde for 30 min and washed with PBS buffer three times. Then the cells were added with 0.1% Triton-100 for 10 min to break the cell membrane and washed with PBS buffer three times. Goat serum blocking solution (5%) was added and discard it after blocking for 30 min. The cells were directly added with anti-eMyHC (1:50, Santa Cruz, SC-53091, Mouse) and incubated overnight at 4°C. The cells were washed again with PBS buffer three times. The secondary antibody anti-mouse (1:150, CWBIO, CW0102S, China) was added, and the culture was placed in a constant-temperature incubator at 37°C for 1 h. Then the cells were washed with PBS buffer three times. Sealing with anti-fluorescence attenuation sealing tablets containing DAPI (Solarbio, S2110, China), and taking pictures under a microscope.

The following antibodies were used: chicken anti-GFP 1:1000 (#ab13970, Abcam), mouse anti-KI67 1:80 (#556003, BD Pharmingen), mouse anti-MYOD 1:80 (M3512, DAKO), rabbit anti-MYOD 1:1000 (sc304, Santa Cruz), mouse anti-MYOGENIN 1:100 (F5D-c, DSHB), mouse anti-embryonic MyHC 1:50 (F1.652, DSHB), mouse anti-embryonic MyHC 1:300 (sc53091, Santa Cruz), mouse anti-MyHC 1:100 (mf20-c, DSHB), rabbit AlexaFluor647-conjugated anti-laminin 1:200 (NB300-144AF647, Novus Biological), rabbit anti-laminin 1:400 (L9393, Sigma Aldrich), rat anti-laminin 1:1000 (4H8-2, Sigma-Aldrich), mouse anti-PAX7 1:100 (Pax7-c, DSHB), rabbit anti-CDKN1c 1:100 (sc8298, Santa Cruz), goat anti-CDKN1c 1:50 (sc1039, Santa Cruz), AlexaFluor-coupled secondary antibodies (Life Technologies, Jackson ImmunoResearch).