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Fig. 5. Src family kinases are required for Mer-mediated downstream tyrosine phosphorylation. (A) Challenge of Mer-expressing cells with apoptotic cells. Mer-expressing cells starved for 18 hours, were co-cultured with apoptotic T cells (AC) at a ratio of 1:10 (Mer-expressing cells: apoptotic cells). Mer was immunoprecipitated from extracts for subsequent in vitro kinase assay. The arrow represents Mer. (B) Mer-mediated FAK^Tyr861 and p130^CAS phosphorylation is dependent on SFKs. Control NIH3T3 cells and SYF cells were transfected with empty vector or CDMer. Cell lysates were analyzed by immunoblotting with anti-phosphotyrosine mAb, and with anti-Mertk Ab to show equal Mer protein recovered (i). Kinase activity of active CDMer immunoprecipitated with anti-Mertk Ab from NIH 3T3 and SYF cells was compared by in vitro kinase assay to show Mer kinase is functional (ii). (C,D) Cell lysates were immunoblotted with anti-phospho-FAK at Tyr⁸⁶¹ (C), or immunoprecipitated with anti-p130^CAS antibody to show the level of tyrosine phosphorylation (D). (E) Mouse NIH3T3 or SYF cells were expressed with pCx-IRES-EGFP or pCx-IRES-EGFP-ß5 using retrovirus infection method as described in the Materials and Methods. When coexpressed with or without Mer, the NIH3T3 and SYF cells were analyzed for phagocytosis assay as described in the legend for Fig. 4. As indicated, phagocytotic ability of NIH3T3 or SYF cells expressing control vector pCx-IRES-EGFP was arbitrarily set as 100% (grey) with cells coexpressing ß5 and Mer shown in black.

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