Anti myhc

Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with phosphate-buffered saline with Tween 20 (PBST) containing 5% fat-free milk, PVDF membranes were coincubated with the antibodies: anti-MyoD (1:1000, abcam, ab16148, Cambridge, UK); anti-MyHC (1:10,000, sigma M4276, Burlington, MA, USA); and anti-GAPDH (1:10,000, Abcam, ab181602, UK) at 4 °C overnight. Subsequently, PVDF membranes were incubated with the corresponding secondary antibody at 37 °C for 1 h. Proteins were detected using the ECL kit (Beyotime, Beijing), and visualized using a Tanon-5200 Multi (Tanon, Shanghai, China) device. Densitometry analysis was performed using the Image J software (Version 1.8.0).The original picture of western blots in this study can be found in Supplementary Material (Figure S1).

C2C12 or HSMM cells were grown on coverslips (SPL Life sciences, Phocheon-si, Korea, Cat#20018). To evaluate the protective effects of CCL2 or hCCL2 on dexamethasone-induced muscle atrophy, differentiated MTs were cotreated with recombinant CCL2 or hCCL2 (100, 200 ng/ml) and dexamethasone (200 μm) for 24 hours. C2C12 or HSMM MBs and MTs were fixed in ice-cold 4% PFA for 15 min, washed three times with PBS, and incubated in ice-cold 0.25% Triton X-100 at room temperature for 10 min [49 (link)]. Then, cells were blocked with blocking solution (1% BSA in PBS) and washed three more times. Next, cells were incubated overnight in anti-MyHC (1:400, Sigma Aldrich, St. Louis, MO, USA) at 4° C. Following three washes in PBS, the cells were incubated with Alexa 555–labeled anti-mouse IgG antibodies (1:1000, Cell Signaling, MA, USA) for 60 min. Then, cells were stained with 4, 6-diamidino-2-phenyindole (DAPI, 1:5000, Sigma Aldrich, St. Louis, MO, USA) for 10 min and washed with PBS three times. The images were produced using an LMS800 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The fusion index (%) was calculated using the following equation: 100×number of nuclei in MyHC+ MTs per total number of nuclei in MyHC+ myocytes and MTs.

The total protein of cells was lysed in a RIPA buffer containing 1% protease inhibitors (PMSF) and collected. After SDS-PAGE, the proteins were transferred onto polyvinylidene fluoride membranes. The primary antibodies were anti-PKNOX2 (Proteintech, Wuhan, China), anti-MyoG (Abcam, Cambridge, UK), anti-MEF2C (Proteintech, Wuhan, China), anti-MyHC (Millipore, Billerica, MA, USA), and anti-β-tubulin (Servicebio, Wuhan, China). The HRP-conjugated secondary antibodies HRP-labeled goat anti-mouse IgG (Servicebio, Wuhan, China) and HRP-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China) were used.

RMS cells were seeded overnight in 6-well plates. Cells were treated with 5-aza-dC or transfected with miRNA mimics for 72–96 h and lysed with Hepes buffer (20 mM Hepes, 250 mM NaCl, 0.1% Triton X-100, 2 mM EDTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 4 mM sodium orthovanadate, 1 mM DTT). Total protein extracts (30 μg) were separated on 8-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA). Filters were blocked with 5% non-fat dry milk in PBS-Tween for 30 min at RT and incubated with the primary antibody. The following antibodies were incubated over-night at +4°C: anti-IGFR1 (Cell Signaling), anti-phospho-Akt (Cell Signaling), anti-MyoR(Santa Cruz Biotechnology), anti-MyOD1 (Millipore), anti-Myf5 (Millipore), anti-desmin (Millipore) and anti-MyHC (Millipore). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) were used for 1 h at RT. Protein-antibody complexes were detected with ECL Super Signal (Pierce). Tubulin (Sigma-Aldrich) was used as a normalization control for equal loading. Experiments were performed at least three times.

Antibodies used for flow cytometry were AF700-anti-mouse Sca-1 (Thermo, 56-5981-82), PerCP/Cy5.5-anti-mouse CD11b (BD Biosciences, 550933), PerCP/Cy5.5-anti-mouse CD31 (BD Biosciences, 562861), PerCP/Cy5.5-anti-mouse CD45 (BD Biosciences, 550944), FITC anti-mouse CD34 (BD Biosciences, 553733), APC-anti-Integrin a7+ (R&D, FAB3518A).
Antibodies used for western blots were Myogenin (F50D) (Santa Cruz, SC-12732), Flag-Tag (3B9) mAb (Abmart, M20008), GAPDH (14C10) Rabbit mAb (Cell Signaling, #2118), Goat anti-mouse IgG-HRP (Santa Cruz, SC-2005), Goat anti-rabbit IgG-HRP (Santa Cruz, SC-2004).
Antibodies used for immunofluorescence staining were anti-MyoD (Santa Cruz, sc-377460), anti-Pax7 (DHSB, RRID:AB_528428), anti-Myh3 (DHSB, F1.625), anti-MyHC (Millipore, 05-716), anti-Laminin (Abcam, ab11575), anti-GFP (Aves Labs, GFP-1010).
Antibodies used for ChIP assays were anti-H3K4me1 (Abcam, ab8895), anti-H3K27ac (Abcam, ab4729), HA antibody (generated by our lab), mouse IgG (Abmart, B30010M), rabbit IgG (Abmart, B30011M).