Anti lrp5

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).

Total protein was extracted from cells using RIPA lysis buffer (Sangon Biotech Co., Ltd.) and quantified using a BCA protein kit (Sangon Biotech Co., Ltd.). Equal amounts of protein (20 µg/lane) were separated via 10% SDS-PAGE and transferred onto PVDF membranes, which were blocked in TBST (0.1% Tween-20) with 5% non-fat milk for 1 h at room temperature. Subsequently, the membranes were incubated with the following primary antibodies overnight at 4˚C: anti-Ckip-1 (1:500; cat. no. D122120; Sangon Biotech Co., Ltd.), anti-β-actin (1:3,000; cat. no. 4970; Cell Signaling Technology, Inc.), anti-Lrp5 (1:1,000; cat. no. ab38311; Abcam), anti-β-catenin (1:1,000; cat. no. 8480; Cell Signaling Technology, Inc.) and anti-α-tubulin (1:2,000; cat. no. 2125; Cell Signaling Technology, Inc.). Following primary incubation, the membranes were incubated with a fluorescein-conjugated secondary antibody (1:3,000; cat. no. ab150079; Abcam) for 2 h at room temperature. Protein bands were visualized using ECL reagent (Sangon Biotech Co., Ltd.) and Odyssey V3.0 image scanning (LI-COR Biosciences).