Iqgap1

A set of three shRNAs against IQGAP1 and controls (Santa Cruz Biotech), were used following the manufacturer’s protocol as previously described [24 (link), 27 (link), 45 (link)]. After 48 h, the cells were counted for proliferation or lysed and protein depletion was evaluated by immunoblotting as described above.

Sixty microlitres of Protein G magnetic beads (1614023, Bio-Rad) were incubated with antibodies for 2 h at room temperature. Then, the protein extracts were added to the beads and incubated overnight at 4 °C with rotation. The beads were washed with 1 × PBST. The proteins bound to the beads were eluted in standard 1 × SDS buffer and heated at 90 °C for 10 min. Finally, proteins were electrophoresed on 10% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membrane for the immunoblot analysis. IQGAP1 (sc-376021, Santa Cruz; 1:50) and PAFAH1B1 (sc-374586, Santa Cruz; 1:50) were used as the precipitating antibodies.

Cell lysates and immunoprecipitated samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 3% skim milk or 5% bovine serum albumin in Tris-buffered saline containing Tween 20. The resulting membranes were then incubated with primary antibodies against β-catenin/catenin beta-1 (E-5, Santa Cruz Biotechnology), PTGS2/COX-2 (D5H5, Cell Signaling Technology), FLAG tag (M2, Sigma-Aldrich), GAPDH (1E6D9, Proteintech), HA tag (3F10, Roche), IQGAP1 (H109, Santa Cruz Biotechnology), JNK (#9252, Cell Signaling Technology), phospho-SAPK/JNK (81E11, Cell Signaling Technology), Lamin B1 (3C10G12, Proteintech), Myc tag (9B11, Cell Signaling Technology), ROBO4 (AF2366, R&D Systems), TRAF7 (H-300, Santa Cruz Biotechnology), mono- and poly-ubiquitinylated conjugates (FK2, Enzo Life Sciences), and RAC1 (PA1-091, Thermo Fisher Scientific) and secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were detected using an ImageQuant LAS4010 system (GE Healthcare). The primary antibodies used are listed in Table S3.