Tgfb2

Total lysates from heart tissues or cells were prepared using lysis buffer (Cell Signaling). Tissues/cells were crushed using beads for homogenization and were quantified. For the detection of the protein following antibodies were used β-catenin, RUNX2 (cell signaling), TGFB2 (Santacruz), MMP2 (abcam), β-ACTIN (sigma) and ARKR1B3 (GeneTex). Antibodies were used at a final concentration of 1μg/ml.

Total protein was isolated from camel tissues using lysis buffer (Solarbio, Beijing, China) and quantified using a BCA protein assay kit (Boster). Samples equivalent to 50 μg of protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were electrotransferred to PVDF membranes (Millipore CAT, Billerica, MA, USA). The membranes were blocked for 2 h in Tris–HCl buffer containing 5% (w/v) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech). Membranes were then washed three times with PBS containing 0.1% Tween 20, incubated with goat anti-mouse/anti-rabbit IgG conjugated to horseradish peroxidase for 2 h at room temperature (1:5000, Bioss, Beijing, China), then washed three times with PBS containing 0.1% Tween 20. All immunoblot assays were performed at least three times. Optical densities of the bands were quantified, and membranes were scanned using Image-Pro plus 6.0 (Media Cybernetics Co., Rockville, MD, USA).