Ab64636

Protein expressions in the ipsilateral hemisphere were measured by western blot as previously described [3 (link)]. Protein samples with equal volumes were loaded on an SDS-PAGE gel, and then electrophoresed protein bands were transferred to a nitrocellulose membrane. The membrane was incubated with the following primary antibodies overnight at 4 °C: rabbit polyclonal anti-frizzled 7 (1:1000; ab64636, Abcam, MA), rabbit polyclonal anti-WISP1 (1:2000; ab178547, Abcam, MA), mouse monoclonal anti-Dvl (1:200; sc-166303, Santa Cruz Biotechnology, TX), rabbit monoclonal anti-β-Catenin (1:10,000; ab32572, Abcam, MA), rabbit polyclonal anti-phospho-β-Catenin (Ser33/37/Thr41) (1:1000; #9561, Cell Signaling, MA), rat monoclonal anti-ZO-1 (1:200, sc-33725, Santa Cruz Biotechnology, TX), rabbit polyclonal anti-Claudin-5 (1:500; ab15106, Abcam, MA), and mouse monoclonal anti-VE-Cadherin (1:500; sc-9989, Santa Cruz Biotechnology, TX). Appropriate secondary antibody (Santa Cruz Biotechnology, TX) was applied to the membranes and incubated for 1 h at room temperature. Immunoblots were probed and exposed to films. Band densities were analyzed using Image J software (NIH, Bethesda, MD).

The following plasmids were used: human DVL2–GFP (pEGFP-N1); SNAP–FZD5 (Koo et al., 2012 (link)); LRP6–GFP (Metcalfe et al., 2010 (link)); PX330-spCas9 (Addgene, #43330) for DVL TKO; PX458-spCas9-GFP (Addgene, #48138) for LRP6 KO. DVL2–GFP was subcloned into pBabe PURO (Addgene) to generate stable cell lines. DVL2 mutants were generated by standard procedures and verified by sequencing. The following antibodies were used: anti-tubulin (1:5000; T4026, Sigma), anti-GFP (1:5000; G1544, Sigma), anti-actin (1:5000; ab8227, Abcam), anti-FZD3 (1:2500; ab75233, Abcam), anti-FZD6 (1:5000; ab128916, Abcam), anti-FZD7 (1:1000; ab64636, Abcam), anti-FZD8 (1:3000; ab40012, Abcam), anti-SNAP (1:5000; P9310S, NEB), anti-DVL1 (1:1000; sc8025, Santa Cruz), anti-DVL3 (1:1000; sc26506, Santa Cruz), anti-DVL2 (1:5000; 3216, Cell Signaling), anti-ABC (1:5000; 8814, Cell Signaling), anti-p-LRP6 S1490 (1:2500; 2568, Cell Signaling).

Total cellular proteins were extracted using M-PER™ Mammalian Protein Extraction Reagent (78501, Thermo) and phenylmethanesulfonyl fluoride. Lysate protein concentrations were quantified with a Bicinchoninic Acid Protein Assay Kit (P8340, Thermo) Twenty micrograms of the protein was separated by 4–12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). Before antibody incubation, PVDF membranes were blocked with 5% non-fat milk solution in TBS with 0.05% Tween-20 at room temperature for 1 h. The membranes were hybridized individually overnight at 4 °C with antibodies in the Cell Cycle Regulation Antibody Sampler Kit (Cell Signaling Technology) and those recognizing GIPC2 (ab175272, Abcam), β-catenin (ab32572, Abcam), Fzd7 (ab64636, Abcam), and ACTB (3700T, Cell Signaling Technology). The blots were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Millipore) for 1 h at room temperature and detected by Super Signal West Pico Chemiluminescent Substrate (Pierce) using an LAS-3000 mini-imaging system (Fuji Film). The signal intensity of each antibody was quantified using Image J software. All results are presented as the mean ± SD (*p < 0.05, n = 3).