Ab86695

Freshly sampled human tissues, mouse tissues or cells were flushed with ice-cold PBS and fixed by incubation in 4% paraformaldehyde in PBS overnight at 4 °C. Fixed samples were dehydrated, embedded in paraffin, and sectioned. The sections were de-waxed and rehydrated. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Antigen retrieval was performed by boiling for 15 min in citrate buffer pH 6.0 or Tris-EDTA pH 9.0. Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology). Staining was performed using the Zsbio kit (Beijing, China) according to the manufacturer's instructions.

Western blots were performed using standard protocols in 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Primary antibody and dilutions used were the following: mouse monoclonal anti-HA (11 583 816 001, Roche, Mannheim, Germany); rabbit polyclonal anti-NIS (24324-1-AP, Protein-tech, Rosemont, IL, USA); mouse monoclonal anti-PCNA (NA03, Merck, Darmstadt, Germany); rabbit anti-GLUT1 (ab652; Abcam, Cambridge, UK); goat anti-GLUT1 (sc-1603; Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-RAC1 (05-389, Millipore, Burlington, MA, USA); mouse monoclonal anti-SRC antibody (60315-1-lg, Proteintech, Rosemont, IL, USA); rabbit anti-phospho Y416-SRC antibody D49G4 (#6943, Cell Signaling, Danvers, MA, USA); mouse monoclonal anti-VAV2 antibody (sc-271442, Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-phospho Y172-VAV2 antibody (ab86695, Abcam, Cambridge, UK); mouse monoclonal anti-P120 catenin antibody 15D2 (#33-9600, Thermofisher, Waltham, MA, USA); rabbit polyclonal anti-phospho Y228-P120 catenin (329050; US Biological, Salem, MA, USA). HRP-labeled anti-rabbit or anti-mouse IgG secondary antibodies (Bio-Rad, Hercules, CA, USA) were used for chemiluminescence imaging.