Ab175272

Western blots of TCLs were performed with rabbit antibodies for GIPC1 (Proteintech Group; 14822–1-AP) and GIPC2 (Abcam #ab175272). A mouse antibody was used for PLXND1 (R and D Systems #MAB41601). GAPDH (loading control) was detected with a rabbit antibody (CST; #5174P).

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).