CARD9 links Ras-GRF1 to H-Ras in response to C. albicans yeast stimulation. (A) RAW264.7 cells stably expressing Flag-CARD9 were stimulated with UV-inactivated WT or mnn5 yeast cells for indicated times. Cell lysates (Ly) were immunoprecipitated (IP) with anti-Flag antibody. (B) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells for indicated times. Cell lysates were immunoprecipitated with α-CARD9 antibody. The cell lysate or immunoprecipitates were subjected to immunoblots using indicated antibodies. (C and D) HEK293T cells were transfected with expression vectors encoding Ras-GRF1, Myc-H-Ras, and Flag-CARD9 at different combinations. Cell lysates were subjected to immunoprecipitation with anti-Flag (C) or anti-Ras-GRF1 antibody (D). (E) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells for indicated times. Cell lysates were immunoprecipitated with anti–H-ras antibody. Immunoprecipitated (IP) and lysate (Ly) fractions were analyzed by immunoblotting using the indicated antibodies. (F) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells or EGF (as a control) for indicated times. Ras activation was determined by pull-down assay using a Ras activation assay Biochem kit according to the manufacturer’s instructions. Immunoassay of the total and activated Ras was performed for determining the Ras activation. Data shown are representative of three independent and reproducible experiments.