P rac1 cdc42

Analytical 12% SDS-PAGE was performed, and 30 μg of protein were analyzed for each condition, unless otherwise stated. For immunoblotting, proteins in the SDS gels were transferred to a polyvinylidene difluoride membrane using an electroblot apparatus. Antibodies against human fibronectin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), FAK (Cell Signaling Technology, Danvers, MA), p-FAK (Epitomics, Burlingame, CA, USA), N-cadherin (Epitomics, Burlingame, CA, USA), E-cadherin (Epitomics, Burlingame, CA, USA), Snail1 (bs-1371R; Bioss, Boston, MA, USA), COX-2 (Lab Vision Corp., Fremont, CA), c-Jun (Santa Cruz Biotechnology), AKT and p-AKT (both from Cell Signaling Technology, Danvers, MA), p-Rac1/cdc42 (Cell Signaling Technology), and α-tubulin and β-actin (both from Sigma-Aldrich) were used as the primary antibodies. Mouse or rabbit IgG antibodies coupled to horseradish peroxidase were used as secondary antibodies. An enhanced chemiluminescence kit (Supersignal West Pico Chemiluminescence kit; Pierce, Rockford, IL) was used for detection. The FAK inhibitor Y15 was purchased from Sigma-Aldrich (St. Louis, MO).

For glypicans analysis, fibroblasts were treated with 5 mU/ml heparinase III in a serum-free medium for 1 h at 37°C. Zebrafish larvae were homogenized and treated with 25 mU heparinase III in H buffer (20 mM Tris-HCl, pH 7.0, 0.1 mg/ml BSA, and 4 mM CaCl₂) for 1.5 h at 37°C. Samples were harvested using SDS lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich) as previously described (Ferreira et al., 2018 (link)). For analysis of PCP pathway components, 2 × 10⁴ fibroblast cells were seeded in six-well plates and harvested after 2 days using SDS lysis buffer. Equal amounts of denatured proteins were separated via SDS–polyacrylamide gel electrophoresis followed by transfer and antibody inoculation as described previously (Tambe et al., 2019 (link)). Antibodies used were Δ-heparan sulfate (AMSBIO, F69-3G10), chondroitin 6 sulfate (Millipore, MAB2035), GAPDH (Invitrogen, MA5-15738), WNT4 (R&D, MAB4751), mCherry (Rockland, 600-401-P16), COG4 (provided by Dr. Daniel Ungar, University of York, United Kingdom), β-catenin (Santa Cruz, sc-7963), JNK (sc-7345), pJNK (sc-293136), and p-Rac1/cdc42 (Cell Signaling, #2461).