FIGURE 2.

Inhibition of GAS6 and PROS1 activities by soluble TAM receptors. A and B, soluble mTAM receptors composed of either two FNIII and two Ig domains (marked as sTAM) or two Ig domains only (marked as TAM-Igs) are schematically depicted and were produced in mammalian or insect cells, respectively. Conditioned media containing sTAMs (5 μl) or 50 ng of TAM-Igs were treated with PNGaseF for 2 h and then subjected to SDS-PAGE. Soluble mTAM receptors were immunoblotted with Abs against FLAG-tag (sTAM, A) or against His-Tag (TAM-Igs, B). C, mTAM-Igs (10 nm) were incubated with conditioned media containing 50 nm γ-carboxylated hGAS6 (K) or non-carboxylated hGAS6 (W). The hGAS6:mTAM-Igs complexes were pulled down by cobalt resin, resolved by SDS-PAGE and immunoblotted with Abs against GAS6 (non-reducing condition, top panels) or His-tag (reducing condition, bottom panels). Beads-alone lane showed non-carboxylated GAS6 weakly bound nonspecifically to cobalt resin (W lane on the GAS6 pull down, long exposure panel). One-twentieth of total reaction volume was used for input control (left two panels). Right two panels show results of pull down experiments. The short and long exposure films were both displayed for anti-GAS6 results. D–F, conditioned media containing soluble mTAMs (50 μl, D) or mTAM-Igs (500 nm, D–F) were mixed with 50 nm of hGAS6 (D) or hPROS1 (E and F), or 20% FBS (F) and incubated at 22 °C for 30 min. Serum-starved mAXL/γR1 (D), mTYRO3/γR1 (E), and mMER/γR1 (F) reporter cells were then treated with the mixtures as indicated on the figure for 30 min at 37 °C, and the activation of STAT1 was detected by pSTAT1 immunoblotting. Immunoblotting results are representative results of three independent experiments.