Figure 5. Dectin-1/Syk stimulation by C.... | Rockefeller University Press

Figure 5.

$Figure 5. Dectin-1/Syk stimulation by C. albicans yeast induces the formation of a Ras-GRF1–CARD9–H-Ras complex. (A) WT and Dectin-1–deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells (MOI = 5) for the indicated times. Cell lysates were immunoprecipitated with anti-CARD9 antibody and immunoprecipitated (IP) and lysate (Ly) fractions were analyzed by immunoblotting using indicated antibodies. (B) WT and Dectin-1–deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells (MOI = 5) or EGF for indicated times. Ras activation was determined by pull-down assay using a Ras activation assay Biochem kit. Immunoassay of the total and activated Ras was performed for determining the Ras activation. (C) RAW264.7 cells stably expressing Flag-Card9 were pretreated with or without Syk inhibitor (piceatannol) for 30 min before stimulation with UV-inactivated mnn5 yeast cells (MOI = 5) for indicated times. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting using indicated antibodies. (D) WT BMDMs were pretreated with or without piceatannol for 30 min before stimulation with UV-inactivated mnn5 yeast cells (MOI = 5) for the indicated times. (E) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells (MOI = 5) for the indicated times. (F) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells for indicated times. Cell lysates were immunoprecipitated with anti-Ras-GRF1 antibody and immunoprecipitated (IP) and lysate (Ly) fractions were analyzed by immunoblotting using indicated antibodies. (G) WT and CARD9-deficient BMDMs were stimulated with plate-coated curdlan (50 µg/ml) for the indicated times. (H and I) Knockdown of endogenous H-Ras by RNA interference in BMDMs, which were transfected with siRNA against murine H-Ras and nontargeting control siRNA using Trans IT-TKO transfection reagent (Mirus). Cells were cultured for 48 h after transfection and then stimulated with plate-coated curdlan (50 µg/ml), UV-inactivated mnn5 yeast cells (MOI = 5), plate-coated α-mannans (50 µg/ml), or LPS (100ng/ml) for the indicated times. Cell lysates were subjected to immunoblotting analysis using indicated antibodies. Data shown are representative of three independent and reproducible experiments.$

Dectin-1/Syk stimulation by C. albicans yeast induces the formation of a Ras-GRF1–CARD9–H-Ras complex. (A) WT and Dectin-1–deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells (MOI = 5) for the indicated times. Cell lysates were immunoprecipitated with anti-CARD9 antibody and immunoprecipitated (IP) and lysate (Ly) fractions were analyzed by immunoblotting using indicated antibodies. (B) WT and Dectin-1–deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells (MOI = 5) or EGF for indicated times. Ras activation was determined by pull-down assay using a Ras activation assay Biochem kit. Immunoassay of the total and activated Ras was performed for determining the Ras activation. (C) RAW264.7 cells stably expressing Flag-Card9 were pretreated with or without Syk inhibitor (piceatannol) for 30 min before stimulation with UV-inactivated mnn5 yeast cells (MOI = 5) for indicated times. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting using indicated antibodies. (D) WT BMDMs were pretreated with or without piceatannol for 30 min before stimulation with UV-inactivated mnn5 yeast cells (MOI = 5) for the indicated times. (E) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells (MOI = 5) for the indicated times. (F) WT and CARD9-deficient BMDMs were stimulated with UV-inactivated mnn5 yeast cells for indicated times. Cell lysates were immunoprecipitated with anti-Ras-GRF1 antibody and immunoprecipitated (IP) and lysate (Ly) fractions were analyzed by immunoblotting using indicated antibodies. (G) WT and CARD9-deficient BMDMs were stimulated with plate-coated curdlan (50 µg/ml) for the indicated times. (H and I) Knockdown of endogenous H-Ras by RNA interference in BMDMs, which were transfected with siRNA against murine H-Ras and nontargeting control siRNA using Trans IT-TKO transfection reagent (Mirus). Cells were cultured for 48 h after transfection and then stimulated with plate-coated curdlan (50 µg/ml), UV-inactivated mnn5 yeast cells (MOI = 5), plate-coated α-mannans (50 µg/ml), or LPS (100ng/ml) for the indicated times. Cell lysates were subjected to immunoblotting analysis using indicated antibodies. Data shown are representative of three independent and reproducible experiments.

This Feature Is Available To Subscribers Only