(A) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for tagged ubiquitin, SUMO1, SUMO2, SUMO3, and ISG15. (B) WT BDMCs were stimulated with depleted zymosan and immunoprecipitated for CARD9, then blotted for endogenous CARD9 and ubiquitin. (C) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for ubiquitin in the presence of either WT or a ligase-dead (C11A,C14A) version of TRIM62. (D) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for tagged ubiquitin. (E–F) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for indicated ubiquitin mutants. In (E), transfected ubiquitin constructs have single point mutations to indicated lysine residues. In (F), transfected ubiquitin constructs have point mutations to multiple lysines, leaving only the indicated lysine residue intact. FS, FLAG-StrepII. See also Figure S4.