Phospho c kit

HEK293, K562 and M0E7 cells and P9 or 3 month-old mouse testes were homogenized in 1X RIPA buffer (Millipore) supplemented with protease inhibitors (complete mini, Roche) with rotation at 4°C for 30 min. Cell lysates were centrifuged at 15,000 g for 20 min at 4°C. Cell debris were discarded, protein amount quantified and co-immunoprecipitations were performed overnight at 4°C using anti-c-Kit (Santa Cruz) or anti-HA antibodies (Abcam) and specific Ig isotypes as negative controls. Pre-blocked protein G beads were used to pull-down the protein complexes (Trueblot).
WGA pull-down experiments were performed as previously reported [47 (link)]. Antibodies were: goat anti-c-Kit (Santa Cruz; R&D) for mouse experiments; anti-c-Kit mouse monoclonal antibody (E-3) against the c-terminus of human c-kit (Santa Cruz) for the human line experiments, Phospho-c-kit (Tyr703; Cell Signaling), rabbit anti-RanBPM (Abcam), anti-HA-Peroxidase high affinity (Roche), anti-pAkt, anti-pErk (Cell signaling), anti-β-actin-HRP, anti-MVH (Abcam), anti-GAPDH mouse monoclonal against rabbit muscle GAPDH (Millipore, MAB374).

Frozen tumor samples were homogenized in a RIPA buffer containing phosphatase and protease inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, ThermoFisher) and immunoblotted. ImageJ (available online: https://imagej.nih.gov/ij/) was used for signal quantification and relative quantification was calculated in comparison with β-Actin. The following primary antibodies were used: CD117 (A4502, Agilent Technologies Dako), Phospho-c-KIT (#3391, Cell Signaling, Leiden, The Netherlands), β-Actin (A1978, Sigma-Aldrich, Milan, Italy).