Fig 2: AXL/MER inhibitors reduce GAS6-stimulated pAKT signal in a concentration-dependent manner. a Representative images of phosphorylated AKT (pAKT) with and without growth arrest–specific protein 6 (GAS6) stimulation. pAKT in G361 cells shows dose response to AXL/MER kinase inhibitors. Green fluorescence represents cells from individual wells expressing pAKT, stained using primary anti-pAKT antibody followed by fluorescein isothiocyanate conjugated secondary antibody. b Nonlinear regression analysis of inhibitor concentration and pAKT response for 4 AXL/MER kinase inhibitors. Results are shown as mean ± standard deviation (SD) of 3 wells. DMSO: dimethyl sulfoxide; IC50: half-maximal inhibitory concentration

Fig 3: IC50 for pAKT inhibition in G361 cells and pMER inhibition in MER overexpressed Ba/F3 cells. a Nonlinear regression analysis of AXL/MER inhibitor concentration and phosphorylated MER (pMER) inhibition in MER overexpressed Ba/F3 cells (left panel), and pAKT inhibition in G361 cells (right panel). Results are shown as mean ± standard deviation (SD) of 3 wells. b Comparison of cellular half-maximal inhibitory concentration (IC50) for pMER inhibition in MER overexpressed Ba/F3 cells and pAKT inhibition in G361 cells. c Correlation between pAKT inhibition in growth arrest–specific protein 6 (GAS6)-treated G361 cells and pMER inhibition in MER-overexpressed Ba/F3 cells. ELISA: enzyme-linked immunosorbent assay

Fig 4: Proposed model of GAS6-inducible pAKT as a biomarker for MER kinase in G361 cells. EGF(R): epidermal growth factor receptor; FNIII: fibronectin type III; GAS6: growth arrest–specific protein 6; Gla: γ-carboxyglutamic acid; Ig: immunoglobin; LG: laminin G; PDGFR: platelet-derived growth factor receptor; RTK: receptor tyrosine kinase; SHBG: sex hormone–binding globulin

Fig 5: GAS6 induces pAKT in human melanoma G361 cells. a Western blot showing the expression of AXL, MER, and TYRO3 among 9 different human cancer cell lines. b and c High-content imaging assay showing robust phosphorylated AKT (pAKT) activation upon growth arrest–specific protein 6 (GAS6) stimulation. GAS6-activated pAKT can be reversed by AXL/MER inhibitor 3 in G361 cells. Three independent experiments were conducted. Results are shown as mean ± standard deviation (SD) of 3 wells from a representative experiment (*p < 0.05)