Profiling Phospho-Kinase Signaling in Cancer Cells

Human phospho-kinase array kits (ARY003B, R&D Systems, Minneapolis, MN, USA) were blotted as per manufacturer's protocol. HCC38 cells were sorted as previously described [7 (link)]. Whole cell lysates were prepared with RIPA lysis buffer (100 mM Tris pH 7.5, 150 mM sodium chloride, 0.1% deoxycholate, 0.1% SDS, 50 mM NaF, Protease inhibitor cocktail (Roche), 2 mM PMSF, 2 mM sodium orthovanadate) combined with scraping and the lysates cleared by centrifugation. Standard Western blotting procedures were performed. The following primary antibodies were used for immunoblotting at a dilution of 1:1000: AXL (8661, Cell Signaling Technology, Danvers, MA, USA), c-Jun (2315, Cell Signaling Technology), pS63 c-Jun (91,952, Cell Signaling Technology), Hsp90 (sc-13119, Santa Cruz, Dallas, TX, USA), or 1:4000 β-actin (MABT825, MilliporeSigma). Treatments with 5 μM JNK–IN–8 (Selleckchem) or vehicle control (DMSO) were performed for 24 h prior to harvesting lysates. All blots or gels derive from the same experiment and were processed in parallel. See Supplementary Material for unedited blots (Supplementary Figs. S5 and S6).