Anti myf5

Cells were lysed in RIPA lysis buffer (Fdbio science, Hangzhou, China) containing PMSF (Genstar) and then centrifuged at 4°C to collect the supernatant. Proteins were quantified using a BCA Protein Assay Kit (Beyotime, Shanghai, China), and equal samples were separated by 10% SDS‒PAGE and then transferred to PVDF membranes. After being blocked in 4% skimmed milk (Ruishu Biotechnology, Zhengzhou, China), the membranes were incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: anti-MyoD (1:1000; Abcam, Cambridge, UK), anti-Myf5 (1:1000; Abcam), anti-MyoG (1:1000; Abcam), anti-MyHC (1:1000; Abcam), anti-Deltex2 (1:1000; Abcam), anti-Pax7 (1:1000; Abcam), anti-Ub (1:1000; Cell Signaling Technology, Beverly, USA), anti-Flag (1:1000; Cell Signaling Technology), anti-HA (1:1000; Cell Signaling Technology), anti-H3 (1:1000; Cell Signaling Technology), anti-GAPDH (1:5000; Bioworld) and β-tubulin (1:1000; Cell Signaling Technology). After three washes in TBS-Tween 0.1%, the membranes were incubated with the corresponding HRP-conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1 h. Immunoblots were detected using ECL (FDbio Science) and a BioLight system (Guangzhou, China).

At the 3-h time point of HS treatment, cultured BSCs were harvested using 150 µL of NP-40 lysis buffer (Thermo Fisher Scientific) with protein inhibitor (Thermo Fisher Scientific). The protein content of the samples was quantified using the BCA protein assay kit (Thermo Fisher Scientific). For immunoprecipitation (IP), anti-HSP27 (5 µg, DSHB), normal rabbit serum (Invitrogen), and Protein A-Sepharose 4B (Invitrogen) were added to the cell lysate, followed by an overnight incubation at 4 °C. The bead–antibody–antigen complex was then centrifuged at 4 °C for 1 min, and the supernatant was discarded. The complex was washed three times for 1 min each with NP-40 buffer at 4 °C. After washing, 50 µL of sample buffer was added, and the mixture was boiled at 95 °C for 10 min. The samples were subsequently analyzed by western blotting. For western blotting, anti-Myf5 (Abcam) and Goat anti-rabbit IgG H&L (HRP) were used at a dilution of 1:1,000.

Cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100/PBS for another 15 min. Then, the cells were blocked with PBS/5% BSA for 1 h and incubated with primary antibodies in PBS/4% BSA overnight at 4°C. The primary antibodies for immunofluorescence were as follows: anti-Deltex2 (1:200; Abcam), anti-Pax7 (1:200; Abcam), anti-MyoG (1:500; Abcam), anti-MyHC (1:500; Abcam) anti-MyoD (1:200; Abcam), and anti-Myf5 (1:200; Abcam). After three washes with PBS, cells were subjected to incubation with appropriate secondary antibodies (Alexa Fluor 488/555, Cell Signaling Technology). DAPI was used to label nuclei for 5 min. Images were acquired using a fluorescence microscope (Nikon, Tokyo, Japan) and confocal microscope (Leica, Heidelberg, Germany) equipped with×10, ×20 and ×100 magnification objectives.