Tgfbr1

C2C12 cells were seeded at 2 × 10⁵ cells/cm² into a 6 cm dish, and cell lysates were collected after VV stimulation from days 1 to 3. RIPA Lysis and Extraction Buffer (89900, Thermo Fisher Scientific Inc., Waltham, MA, USA) containing protease and phosphatase inhibitor (1:100; A32961, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to extract the cell lysate. Western blot analysis was performed with primary antibodies against stathmin (1:1000), decorin (1:1000), Col-I (1:500), FGF7 (1:1000), TGFBr1 (1:500) and PAK3 (1:500) (Abcam Inc., Cambridge, MA, USA), pAKT (1:500), AKT (1:2000), MyoD (1:1000) and myogenin (1:500) (Cell Signaling Inc., Danvers, MA, USA), β-actin (1:10,000) (Arigo Biolaboratories Corp., TE Huissen, Netherlands), which were incubated with the membranes at 4 °C overnight. After being washed 3 times for 15 min each, the membranes were incubated with an HRP-conjugated goat antibody anti-mouse or anti-rabbit IgG secondary antibody (Arigo Biolaboratories Corp., TE Huissen, Netherlands) for 1 h at RT. The proteins were detected using an ECL substrate kit (Abcam Inc., Cambridge, MA, USA). Band intensity was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Intensity data were normalized to GAPDH and indicated controls for the target protein fold changes calculation.

The experimental procedures for the extraction and detection of protein expression were performed following the protocols mentioned in the previous study [10 (link)]. The detailed information of antibodies were listed as follows: Nanog (Cat No. 14295-1-AP, 1:1000, Proteintech, Wuhan, China), ALG10 (Cat # ab124711, 1:3000, Abcam), Oct4 (Cat No. 11263-1-AP, 1:1000, Proteintech), Sox2 (Cat No. 11064-1-AP1:1000, Proteintech), TGFBR2 (Cat No. 66636-1-Ig, 1:1000, Proteintech), TGFBR1 (Cat # ab31013, 1:1000, Abcam), p-Smad2 (Cat # ab28088, 1:1000, Abcam), Smad2 (Cat No. 12570-1-AP, 1:1000, Proteintech), GAPDH (Cat No. 60004-1-Ig, 1:1000, Proteintech), Histone H3 (Cat No. 17168-1-AP, 1:1000, Proteintech) were used in this study. The full length uncropped original western blots used in their manuscript were shown in Supplementary Figure 1.

Briefly, 20ug total protein was loaded in each lane and transferred to a PVDF membrane (Bio‐Rad). The membrane was then blocked with 5% non‐fat dried milk and incubated overnight at 4°C with TGFBR1(Abcam) and β‐actin (1:2000 dilution; CMCTAG) primary antibody and incubated with horseradish peroxidase (HRP)‐conjugated goat secondary antibodies for 1 hour at room temperature. Protein bands were finally visualized by enhanced chemiluminescence (ECL) and detected using the ECL Detection System (Amersham Pharmacia Biotech).

SDS-PAGE was performed using a Novex 16% Tris-Glycine gel (Life Technologies/Invitrogen, Carlsbad, CA, USA) and a 15% e-PAGEL mini gel (ATTO Corporation, Tokyo, Japan). The gel was stained with Simply Blue Safe Stain (Invitrogen) or ProteoSilver Stain (Sigma-Aldrich). The apparent molecular weights of the protein bands were estimated by comparison with the SeeBlue Plus2 Pre-Stained Standard (Life Technologies/Invitrogen) or the Novex Sharp Protein Standard (Life Technology/Invitrogen). Duplicate gels were transblotted onto Invitrolon polyvinylidene difluoride (PVDF) membranes (Life Technologies/Invitrogen) and immunostained with porcine amelogenin or TGFBR1 (Abcam, Cambridge, UK) polyclonal antibodies. Immunopositive bands were visualized by 3,3′-diaminodbenzidine (DAB) for amelogenin or enhanced chemiluminescence for TGFBR1. Full images of the blots that were cropped in the main figures are shown in Supplementary Fig. S12.