Ab124800

The antibodies used were MyoG (ab124800, Abcam), MyoD (ab133627, Abcam), p-ERK1/2 (9101, Cell Signaling Technology), GAPDH (ab8245, Abcam), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208; Beyotime). Total protein from cultured MuSCs was extracted using the Total Protein Extraction Kit (Beyotime) and quantified using the BCA Protein Quantitation Kit (Beyotime) according to the manufacturer’s instructions. Briefly, proteins (~20 μg per sample) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, Bedford, MA, USA), and blocked with blocking buffer (Beyotime) for 1 h at room temperature. The membranes were sequentially incubated with primary anti-mouse MyoG (1:1000), MyoD (1:1000) and p-ERK1/2 (1:1000), washed three times with PBST (0.1% Tween 20 in PBS), and incubated with the secondary antibody conjugated with HRP (1:4000) for 90 min. The membranes were washed three with PBST. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (BeyoECL Plus, Beyotime) and the ChemiDoc XRS⁺ system (Bio-Rad, Hercules, CA, USA). GAPDH (1:2000) served as a loading control.

Cells were washed with sterile PBS and lysed with ice-cold RIPA buffer containing 1% of protease inhibitors and centrifuged at 10,000 g for 10 min. The supernatant was retained, aliquoted, and the protein content was quantified using the BCA Assay Kit (Thermo scientific, Mississauga, Ontario, Canada). A volume corresponding to 40 μg of protein was diluted with a sample buffer (125 mM Tris buffer (pH 6.8), 4% SDS, 20% glycerol, 0.05% bromophenol blue, and 200 mM dithiothreitol), heated at 100 °C for 5 min and electroseparated on 10% sodium dodecyl sulfate-polyacrylamide gel¹⁰³ . Proteins were transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat milk or 5% BSA for 90 min at room temperature. Membranes were immunoblotted overnight at 4 °C with anti-myogenin (ab124800, 1:500; Abcam) and anti-beta-actin (4967 S, 1:5,000; Cell Signaling) as primary antibodies. After washing, membranes were incubated with goat anti-rabbit (H + L) HRP-conjugated secondary antibody (1:5,000; Abcam, ab6721) for 1 h at room temperature. Bands were revealed with ECL-plus Western blotting reagent (PerkinElmer Life and Analytical Sciences, USA), visualized using G:Box (GeneSys imaging software, v1.8.6.0), and quantified using ImageJ (v1.53, National Institutes of Health, Maryland, USA). Densities were normalized to the loading control.

The anti-CNOT1 mouse monoclonal antibody was described previously [25 (link)]. Antibodies against GAPDH (#2118), PPARγ (#2443), C/EBPβ (#3087), and Perilipin (#9349) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against C/EBPα (sc-61) was from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against UCP1 (ab10983), MyoD1 (ab16148), and Myogenin (ab124800) were from Abcam (Cambridge, UK). The antibody against Myosin (M4276) was from Sigma Aldrich (St. Louis, MO, USA).

Cells were grown on coverslips and fixed for 6 min in 4% paraformaldehyde at room temperature. After washing with PBS, the cells were permeabilized for 6 min using 0.1% Triton X-100, blocked in 1% BSA for 1 h, and incubated for 1 h at room temperature with primary monoclonal antibodies: anti-Nrf2, sc-365949; anti-SQSTM1/p62, sc-28359 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-myogenin, ab124800 (Abcam, Cambridge, MA, USA) diluted in blocking buffer. After washing the cells 3 times with PBS and 2 times with blocking buffer, fluorescently-labeled secondary antibodies diluted in blocking buffer were applied for 30-45 min at room temperature in the dark. The cells were then washed with PBS, and the coverslips mounted on glass slides for microscopy.

Proteins were extracted from the HEK293T transfected with the mixture of equivalent amount of miR-30-5p constructs, using RIPA buffer (Solarbio; Beijing, China) containing 1 mM PMSF (Solarbio; Beijing, China). Then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the 8% or 12% polyacrylamide gels with 20 μg proteins per lane. After this, the proteins in the gels were transferred onto the PVDF membrane that then were blocked with 5% skim milk in Tris Buffered Saline with Tween (TBST) buffer (0.15 M NaCl, 1 M Tris–HCl pH 8.0, 0.05% Tween-20) for 2 h at room temperature. After incubation with primary antibody against MBNL1 (dilution 1:1000; ab108519; Abcam, Shanghai, China), MyoG (dilution 1:1000; ab124800; Abcam, Shanghai, China), MHC (dilution 1:1000; ab24648; Abcam, Shanghai, China) at 4 °C overnight, the PVDF membrane were washed in TBST buffer and incubated with secondary antibody for the anti-immune rabbit IgG (dilution 1:4000; LK2001; Sungene Biotech, Tianjin, China) conjugated with HRP. Antibody reacting bands were detected by means of ECL detection reagents. The β-tubulin was used as internal control, whose primary antibody (dilution 1:1000; KDM9003; Sungene Biotech, Tianjin, China) and secondary antibody for anti-immune mouse IgG-HRP (dilution 1:4000; LK2003; Sungene Biotech, Tianjin, China) were purchased from the Tianjin Sungene Biotech.

Tissues or cells were lysed with RIPA buffer. Protein content in lysates was measured using bicinchoninic acid and equal amounts of proteins (20 μg) were separated using SDS-PAGE with β-Actin as a loading control. Separated proteins were transferred to PVDF membranes (Millipore, USA) and subsequently blocked with 5% nonfat dry milk. The membranes were incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used: CPNE1 (1:1000, abcam, ab155675), ATROGIN1 (1:1000, Abcam, ab168372), MuRF1 (1:1000, Abcam, ab183094), MyoG (1:1000, Abcam, ab124800), MyoD (1:1000, Abcam, ab133627), GLB1 (1:1000, Abcam, ab203749), p-eIF2α (1:1000, Cell Signaling Technologies, #9721), eIF2α (1:1000, Abcam, ab169528), p-PERK (1:1000, Cell Signaling Technologies, #3179), PERK (1:1000, Abcam, ab229912), ATF4 (1:1000, Abcam, ab31390) and β-Actin (1:1000, Abcam, ab8226). Protein gray values were measured using Image J software.