Anti wnt10b

Paraffin sections of the scapulae from wild-type and Wnt5a^−/− embryo at E18.5 were subjected to immunostaining using anti-β-catenin antibodies (R&D systems). Immunocomplexes were visualized with diaminobenzidine (Dako). The sections were counterstained with hematoxylin.
Western blot analyses were performed using anti-GFP (Abcam) and anti-β-catenin (Cell Signaling Technology), anti-β-actin (Sigma), anti-Wnt5a (R&D Systems), Anti-Wnt7b (Santa Cruz), Anti-Wnt10b (Abcam), Anti-Lrp5 (Cell Signaling Technology), anti-Lrp6 (Abcam) antibodies as described previously14 (link). Lysates (40 μg protein/lane) from cells and from scapulae of E18.5 mice embryos were electrophoresed on SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and subjected to immunoblotting using the ECL plus chemiluminescence detection system (GE Healthcare, Buckinghamshire, UK).

Protein samples were collected from cell lysates and protein concentrations were determined using a BCA kit (Beyotime). Proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% Tris-buffered saline with Tween 20 for 2 hours at room temperature and then incubated with primary antibody overnight at 4°C. The primary antibodies were: anti-caspase-3, anti-cleaved caspase-3, anti-caspase-9, anti-cleaved caspase-9, anti-BAX, anti-Bcl-2, anti-CytC, anti-Apaf-1, anti-COX IV, anti-PAX2, anti-GSK-3β, anti-pGSK-3β^(Ser9), anti-β-catenin, anti-p-β-catenin^{(Ser33+Ser37)}, anti-Wnt2, anti-Wnt4, anti-Wnt5a, anti-Wnt10b, anti-Wnt11, anti-Wnt13, anti-Wnt14 (Abcam, Cambridge, UK), anti-GAPDH, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA). Films were cleaned 3 times with TBST and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 hour. Membranes were visualised using the enhanced chemiluminescence reagents (Millipore, Burlington, MA, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).