Syk is required for Cbl-b–mediated ubiquitination and degradation of Dectin-2 and Dectin-3. (A) Hyphae-induced phosphorylation of Cbl-b in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. p-Cbl, phosphorylated Cbl. (B) Hyphae-induced association analyses of Cbl-b with Dectin-2 or Dectin-3 in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. (C and D) Hyphae-induced ubiquitination of Dectin-2 and Dectin-3 in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. (E) Hyphae-induced degradation of Dectin-2 and Dectin-3 in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. (C–E) Cell lysates were immunoprecipitated with the indicated antibodies. (F) HEK293 T cells were transfected with expression vectors encoding Flag–Dectin-2 (D2-Flag), Flag–Dectin-3 (D3-Flag), FcR-γ–HA, Myc-Syk, and Cbl-b in different combinations. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody. The cell lysate and immunoprecipitates were subjected to immunoblots using the indicated antibodies. (G and H) Hyphae-induced ubiquitination of Dectin-2 and Dectin-3 in WT and CARD9-deficient BMDMs. Cell lysates were immunoprecipitated with anti–Dectin-2 or –Dectin-3 antibodies as indicated. The cell lysate and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. The data shown are representative of three independent and reproducible experiments. IP, immunoprecipitation; Ly, lysis; p-Syk, phosphorylated Syke; Ub, ubiquitination.