PI3K is the major signaling pathway downstream to Axl activation. (A) LTC cells were starved overnight in serum-free medium and then stimulated with Gas6-His (1 μg/mL) or Gas6-Fc (1 μg/mL) for 20 minutes. Human Fc fragment (1 μg/mL) was used as a negative control. The whole-cell lysates were either subjected to immunoprecipitation by Axl antibody MAb173, followed by Western blot with Axl antibody (rabbit monoclonal) or 4G10 (biotinylated), or directly subjected to Western blot with antibodies against pAKT or pMAPK. The relative protein level was quantitated by ImageJ, normalized (phosphorylated Axl level was normalized to Axl level; pAKT and pMAPK levels were normalized to β-actin level), and is shown below each panel. (B) LTC cells were transfected by 5nM or 30nM of Axl siRNA and control siRNA. At 72 hours after transfection, whole-cell lysates were subjected to Western blot with antibodies against Axl or pAKT.