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Wnt2b is a secreted signaling protein that is a member of the Wnt family. It plays a role in cell-cell signaling and is involved in various developmental processes.

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2 protocols using wnt2b

1

Immunofluorescence analysis of BMSCs

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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).
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2

Camel Tissue Protein Isolation and Quantification

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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).
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