Ab203383

Cells or EVs were sonicated into the lysis buffer supplemented with phosphatase and protease inhibitors (KGP2100, Keygen Biotech, China). Proteins were transferred onto PVDF membranes. Then the PVDF membranes were blocked by 5% milk (P0216-1500 ​g, Beyotime, China) and incubated with primary antibodies against GAPDH (1:10000, HRP-60004, proteintech, USA), P16 (1:1000, ab51243, abcam, UK), P53 (1:2000, A10610, ABclonal, China), P21 (1:2000, A1483, ABclonal, China), myogenic differentiation antigen (MyoD) (1:100, ab203383, abcam, UK), myogenin (MyoG) (1:200, ab124800, abcam, UK), myosin heavy chain (MyHC) (1:1000, ab91506, abcam, UK), CD9 (1:1000, ab263019, abcam, UK), CD81 (1:1000, ab109201, abcam, UK), TGS101 (1:1000, ab125011, abcam, UK), and Calnexin (1:1000, ab133615, abcam, UK) at 4 ​°C overnight. Membranes were then incubated with goat anti-rabbit IgG(H ​+ ​L) HRP (70-GAR0072, MultiSciences, China) at 37 ​°C for 1 ​h. Subsequently, the immune complexes were visualized using a tanon™ high-sig ECL western blotting substrate (180-5001, Tanon, China) and automatic digital gel/chemiluminescence image analysis system (4600SF, Tanon, China).

The sections were blocked with quickblock blocking buffer for immune staining (P0260, Beyotime, China) for 15 ​min at RT, followed by incubation with primary antibody against Pax7 (1:100, bs-22741R, Bioss, China), MyoD (1:100, ab203383, abcam, UK), MyoG (1:500, ab124800, abcam, UK), fast MyHC (1:1000, ab91506, abcam, UK), laminin (1:50, ab11575, abcam, UK) at 4 ​°C overnight and labeled with Alexa Fluor594-preabsorbed goat anti-rabbit IgG (ab150084, Abcam, 1:500, UK), Alexa Fluor 488 AffiniPure F(ab')₂ Fragment Goat Anti-Rabbit IgG (111-546-003, Jackson ImmunoResearch, 1:500, USA) respectively for 2 ​h at room temperature. Next, the nucleus was stained with DAPI.

Teratomas were homogenized and lysed in RIPA buffer containing protease inhibitors (Complete, Roche), and obtained lysates were kept on ice for 30 min, centrifuged, and stored at − 80 °C. Protein concentration was determined by using Bradford’s assay (Sigma-Aldrich). Twenty microgram protein was subjected to 10% SDS-PAGE and Western blot analysis and probed with antibodies against Pax3 (ARP32446, Aviva; 1:1000), Myf5 (SAB 4501943, Sigma-Aldrich; 1:1000), MyoD (Ab203383, Abcam; 1:1000), Myogenin (sc-576, Santa Cruz Biotechnology; 1:1000), Mck (SAB4500267, Sigma-Aldrich; 1:1000), M-cadherin (SAB4500040, Sigma; 1:1000), or Hsp90 (TA500494, OriGene Technologies). As secondary antibodies, goat anti-rabbit HRP conjugate (170-6515, Bio-Rad) was used, followed by chemiluminescence detection. Films (Amersham Hyperfilm ECL (GE Healthcare)) exposed on membranes were photographed, and optical density of resulting bands was measured using GelDocXR+ (Bio-Rad) with ImageLab software.