Fig 1: C-10 is a selective agonist of TYRO3.(A) Cultured human podocytes were stimulated with C-10 at different doses for 30 minutes. Cell lysates were probed with an antibody recognizing the phosphorylated form of all 3 TAM receptors. Phosphorylated MER, AXL, and TYRO3 receptors are differentiated by their corresponding molecular weight. (B) Immortalized podocytes were transduced with either control vector or lentiviral vectors expressing individual FLAG-tagged TAM receptors (AXL-FLAG, TYRO3-FLAG, or MER-FLAG). Lysates from transduced cells were probed for FLAG-tagged protein expression. (C) Immortalized podocytes from B were treated with 100 nM C-10 for 30 minutes, and lysates were probed for phosphorylated or total AKT (p-AKT or t-AKT) and FLAG proteins. GAPDH was used as a loading control. Representative blots of 3 independent experiments are shown. (D) Drug affinity responsive target stability (DARTS) assay was performed to test the direct binding of C-10 to TYRO3. Podocyte lysates were preincubated with various concentrations of C-10 as indicated at 25°C for 1 hour prior to digestion with Pronase (0 or 1:1000 dilution) for 20 minutes. Lysates were then probed for TYRO3 expression, with GAPDH as a loading control.