AXL kinase activity and the Y779 phosphorylation site are crucial for feedback regulation. (A) Effects of various AXL mutations on AXL 3′-UTR reporter activity. The AXL 3′-UTR reporter construct was cotransfected with various AXL mutants in CL1-3 cells, and luciferase activity was measured 24 h later. (B) Effects of double or triple mutants in AXL tyrosine phosphorylation sites on AXL 3′-UTR reporter activity. Luciferase reporter activity was assayed for CL-3 cells cotransfected with individual AXL mutants and the AXL 3′-UTR reporter construct. The lower panel shows the expression and molecular weights of various AXL mutant proteins verified by Western blot analysis using a mixture of antibodies against the carboxyl and amino terminal of AXL. Soluble AXL was concentrated from the conditioned medium and detected by Western blotting using AXL amino-terminal antibody. Actin was used as a loading control. The values were derived from three independent experiments. Error bars represent SD (*) P < 0.05.
