Figure 4. Gas6 enhances the interaction between glutathiolated MHC-IIB and Axl.

A. Production of H₂O₂ by Gas6. VSMC were loaded with DCF-DA for 30 min and stimulated with either Gas6 (100 ng/ml) or H₂O₂ (300 µM) for 3 min. Relative DCF fluorescence (RDF) was measured for 1 min at 10 sec intervals. Cells were viewed with an Olympus fluorescence microscope. Data are mean±SE for 2 separate experiments. *P<0.01 for increase in RDF versus control. B. VSMC were stimulated with Gas6 (100 ng/ml) for the indicated times. Cell lysates were immunoprecipitated with anti-Axl antibody and immunoblotted with MHC-IIB antibody. Equal loading was confirmed using anti-Axl antibody. C. Cells were loaded with 250 µM BioGEE for 1 hr, stimulated with H₂O₂ or Gas6 for 10 min, and Axl was immunoprecipitated. Non-reducing SDS-PAGE was performed and BioGEE labeled proteins were detected with streptavidin HRP (upper panel). The 225 kDa protein was verified to be MHC-IIB by probing the blot with MHC-IIB antibody, followed by detection using Licor infrared antibodies (middle panel). Lower panel shows immunoprecipitation of Axl.