The soluble ectodomain of Mer binds to Gas6 and inhibits Gas6 signaling. (A) Gas6/Mer complexes in wild-type C57Bl/6 serum were detected by immunoprecipitating with a mouse anti-Gas6 antibody and by performing a Western blotting with an anti–mouse Mer antibody. The Gas6/Mer complexes were not observed in the control serum from C57Bl/6 MerKD mice. (B) A chimeric recombinant protein consisting of the mouse Mer extracellular domain fused to the Fc region of human IgG was incubated with recombinant mouse Gas6 (rmGas6). As a control for nonspecific binding, a parallel experiment was performed with a Ret receptor ectodomain/Fc chimera and mGas6. Complexes pulled down with protein G Sepharose beads were run on SDS-PAGE gels, and bound Gas6 was detected by immunoblotting with anti–mouse Gas6 antibody. Also shown is the input rmGas6. (C) J774 cells were starved in serum-free medium and then treated for 10 minutes with 200 nM mGas6 and 200 nM Mer/Fc or Ret/Fc as indicated. Cell lysates were analyzed for phospho-Mer and total Mer content. (D) J774 cells incubated with or without mGas6 and Mer/Fc as in panel C and phospho-AKT and total AKT were monitored.
