Myosin heavy chain

Cells were homogenized in a radioimmunoprecipitation (RIPA) buffer with protease and phosphatase inhibitors and incubated on ice for 30 min. Samples were then centrifuged at 16,000 RCF for 15 min at 4 °C, and the supernatant was transferred to a new tube.
Protein lysates were resolved using a 4–16% gradient of polyacrylamide gels and transferred to nitrocellulose membranes (Sigma, St. Louis, MO, USA). The following primary antibodies and dilutions were used: myosin heavy chain (1:1000; R&D Systems, Minneapolis, MN, USA), phospho-p65^S536 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), ß-actin (1:10,000; Santa Cruz, Dallas, TX, USA), α-tubulin (1:10,000; Santa Cruz, Dallas, TX, USA), and histone H3 (1:1000; Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated for 1 h at room temperature in species-specific secondary antibodies (LI-COR) diluted 1:5000 in 5% BSA in TBS-T.
Membranes were scanned and densitometry was assessed using the Odyssey infrared fluorescent imaging system using LI-COR software (version 5.2, LI-COR, Lincoln, NE, USA). Raw values were compared between groups only if samples were on the same membrane. Raw values normalized to loading control levels were used to calculate the relative protein levels.

Immunoblot and RT-PCR were performed as described previously13 (link). The following antibodies were used for the western blot analysis: myosin heavy chain (R&D system, Minneapolis, USA), myogenin (Abcam, Cambridge, UK), creatin kinase M (Santa Cruz, California, USA), MET, p-p38, p38, p-AKT, AKT, and α-tubulin (Cell Signaling Technology, Danvers, Massachusetts, USA). PCR condition is as follow: denaturation for 3 min at 94 °C, amplification for 35 cycles of 30 s at 94 °C, 60.7 °C, and 72 °C, final extension for 5 min at 72 °C. The human myogenin primer sequences were: F, 5-AGC GCC CCC TCG TGT ATG-3; R, 5-TGT CCC CGG CAA CTT CAG C-3.

Muscle stem cells on day 4 of culture or day 3 of differentiation were fixed in 4% paraformaldehyde in DPBS for 30 min at 4°C. Fixed cells were washed twice with DPBS and then permeabilized with 0.2% (v/v) Triton X-100 (Sigma-Aldrich, USA) for 15 min before blocking non-specific binding with 10% (v/v) goat serum (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Primary antibodies against chicken Pax7 (1:200; R&D Systems, Minneapolis, MN, USA) or myosin heavy chain (1:1,000; R&D Systems, USA), respectively, were added, and the cells were incubated at 4°C overnight. goat serum and primary antibodies were removed, and cells were incubated with secondary antibodies (1:1,000; Invitrogen, Waltham, MA, USA, A -11001) at 4°C overnight. The nuclei were stained with Hoechst 33342 (1:1,000; Molecular Probes, Eugene, OR, USA) for 10 min at 4°C, and the cells were washed, followed by media replacement with DPBS. Stained images were captured using an inverted fluorescence microscope.