Anti sema7a

Cell pellets were lysed in RIPA buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with complete EDTA-free proteinase inhibitor cocktail tablet (Roche)), and quantified by Bradford assays using the Coomassie Plus Assay Kit (Pierce). Typically, 10 μg total protein was separated on a 12% SDS-PAGE gel, then transferred to nitrocellulose membrane (BioRad). After blocking with 5% milk in 1x TBST, the membrane was probed with the appropriate antibody, detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) according to the manufacturer’s protocol, imaged using a GBox (SYNGENE) and quantified by GeneTools v. 4.02 (SYNGENE). We obtained standard curves for SEMA7A and GRB2 by making serial dilutions of total cell lysate. Both curves show a strong linear relationship between signal and the amount of cell lysate (data not shown). The Western blots presented in the paper were performed within the linear range. Primary antibodies used were anti-SEMA7A (sc-376149, Santa Cruz Biotech), anti-GRB2 (#3972, Cell Signaling), anti-GAPDH (#2118, Cell Signaling) and anti-TUBULIN (#CP06, DM1A, Calbiochem). GAPDH and TUBULIN were provided as normalization controls. Data shown in the figures are representative of at least three independent experiments.