FIGURE 3.

Blocking GAS6 and PROS1 activities by soluble TAM-Igs. mTYRO3/γR1 and mAXL/γR1 reporter cells were starved for 5 h in serum-free media and incubated with serial dilution of soluble mTAM-Igs for 30 min at 22 °C. The size of triangles represent the concentration of TAM-Igs: mTYRO3-Igs (1.43–11.43 μm), mAXL-Igs (10–80 nm), mMER-Igs (1–8 μm) were premixted with AXL/γR1 cells (A); and mTYRO3-Igs (11–714 nm), mAXL-Igs (0.16–10 μm), mMER-Igs (0.13–8 μm) were premixed with mTYRO3/γR1 cells (B). hGAS6 (10 nm, Amgen) and hPROS1 (20 nm) were then added to the cells:TAM-Igs mixture for additional 30 min at 37 °C. The activation of chimeric receptors was assessed by measuring levels of pSTAT1 by immunoblotting, and the levels of pSTAT1 activation induced by ligands alone was set as 100% after normalization to actin loading control. The curve was plotted according to the blocking effects and the concentration of mTAM-Igs. Immunoblotting results are representative results of at least three independent experiments.