Fig. 7. Integrin vß5 is essential for Mer-mediated p130^CAS phosphorylation, Rac1 activation, and phagocytosis. (A) ß5 null CS-1 cells expressing empty vector, ß5 or ß5C were expressed with or without CDMer cells. The lysates were immunoprecipitated with anti-p130^CAS Ab, and analyzed by immunoblotting with anti-phosphotyrosine Ab (i). Alternatively, the lysates were precipitated with GST-PAK CRIB Sepharose beads, and the levels of Rac1 GTP loading were determined by immunoblotting with anti-Rac1 mAb (ii). ß5/ß5C expression was comparable between cells co-overexpressed with and without CDMer, as analyzed with anti-ß5 immunoblotting in RIPA buffer lysates (A, panel i) and P1F6 staining by FACScan (B). Unshaded, control CS-1 cells; shaded, CS-1 cells expressing ß5. (C) Phagocytosis assay. CS-1 cells were transfected with bicistronic pIRES-EGFP, pIRES-EGFP-ß5, or pIRES-EGFP-ß5C, with empty vectors or Mer. After 48 hours, transfected cells were co-cultured with red-labeled apoptotic T cells (1:10), and phagocytosis assay was performed as described in the legend for Fig. 4. Data represent the mean±s.e.m. percentage of phagocytosing cells from four separate experiments (i and ii). (D) CS-1 cells were transfected with bicistronic pIRES-EGFP or pIRES-EGFP-ß5, with control vector (-) or mutant Mer/KD and phagocytosis assay was performed as described above. Data represent the mean±s.e.m. percentage of phagocytosing cells from three separate experiments.