Wnt2b

BMSCs were seeded into 24 well plates with slides and incubated at 37 °C with 5% CO₂ and saturated humidity. 48 hours post-transfection, the cells were fixed in ice-cold 4% paraformaldehyde for 30 min. Permeabilization was performed with 0.25% Triton X-100 in PBS for 10 min followed by blocking for 1h with 10% Goat serum. The fixed cells were then incubated with 1:100 dilution of PIWI (Abcam, UK), β-catenin (Santa Cruz Biotechnology, USA), and Wnt2b (Santa Cruz Biotechnology, USA) overnight at 4 °C. After washing three times with PBS, the slides were incubated with fluorescein isothiocyanate (cy3)-conjugated secondary antibody in a 1:200 dilution in PBS for 1 h at room temperature in darkness. The nuclei were subsequently stained with 4,6-diamidino-phenyindole (DAPI) (Abcam, Cambridge, UK) and the slides were then examined using Leica DMi8 microscope (Carl Zeiss MicroImaging GHBH; Jena, Germany). Statistical fluorescence intensity was performed by using ImageJ as the previous study described 36 (link).

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).