Anti deltex2

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).

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.

Cells were lysed with IP lysis buffer, and the supernatant was obtained by centrifugation. Co-immunoprecipitation was conducted using a Dynabeads™ Protein G Immunoprecipitation Kit (10007D; Invitrogen) according to the manufacturer’s protocol. Briefly, Dynabeads Protein G were washed with Ab Binding & Washing Buffer and then incubated with specific antibody for 30 min. Cell lysates were incubated with the antibody-coated beads overnight at 4°C with rotation. After 3 washes with washing buffer, the immunocomplexes were eluted with elution buffer and subjected to SDS‒PAGE. The antibodies used for IP are listed as follows: anti-Deltex2 (1:50; Abcam) and anti-MyoD (1:50; Abcam).