Fig 1: Western blot-based validation of selected molecules in the Axl signaling pathway. (A). MDA-MB-231 cells were treated with 400 ng/mL GAS6 for the indicated duration. Cell lysates were analyzed with Western blot analysis using phospho-specific antibodies against EPHA2 (pY772), AXL (pY703/pY702), PZR (pY263), SHP2/PTPN11 (pY580), PTPRA (pY798) and CDK1 (pY15) and antibodies against the corresponding protein as indicated. ß-actin served as a loading control. The arrows indicate the three isoforms of PZR protein. (B). Western blot analysis to confirm the dynamic regulation of phosphorylation of the indicated proteins in HCC1395 cells treated with 400 ng/mL GAS6 for different time courses (as indicated). (C). MDA-MB-231 cells were pretreated with 2 µM R428 prior to GAS6 stimulation for 5 or 10 min, respectively. Protein phosphorylation levels were detected by indicated phospho-specific antibodies. (The original western blot images were included in Figure S1).