The AXL ligand GAS6 is crucial for the negative feedback loop. (A) Effects of AXL immunoglobulin domain deletion mutants on AXL 3′-UTR reporter activity. AXL 3′-UTR reporter was cotransfected with wild-type AXL or the immunoglobulin domain deletion mutant into CL1-3 cells, and luciferase activity was measured 24 h later. The expression and molecular weights of various truncated AXL proteins were verified by Western blot analysis (right panel). (B) GAS6 suppresses AXL 3′-UTR reporter activity in a dose-dependent manner. A fixed amount of AXL 3′-UTR reporter construct was cotransfected with various amounts of pcDNA3-AD-Gas6 into CL1-3 cells. The Renilla reporter was used as transfection efficiency control. Expression of GAS6 was concentrated from the conditioned medium and determined by Western blotting (right panel). Actin was used a loading control. (C) GAS6's inhibitory effect on the AXL 3′-UTR reporter activity was neutralized by AXL-Fc. Fixed amounts of the AXL 3′-UTR reporter and the pcDNA3-AD-Gas6 expression construct were cotransfected with various amounts of the AXL-Fc construct into CL1-3 cells. Luciferase activity was measured 24 h post-transfection. Equal amounts of DNA (2 μg) were transfected for each sample in B and C. The values were derived from three independent experiments. Error bars represent SD (*) P < 0.05.
