Gas6 stimulation promotes OPCML-AXL interaction, thereby altering AXL localisation and activation.Figure 2 SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm)  SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course an (B) Pearson"s Correlation R was calculated Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests d; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 (E) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1 Data i M, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests (F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein (G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6 (H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6 (I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation (J) Ratio of AXL band intensity in "R" to total AXL "T" (T = S + R) from panel (I) (K) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests (L) Diffusion Co-efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests (M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests (N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe) Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests (O) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM