Fig. 6.

Ubiquitination of Malt1A K648 by Hectd3 is essential for RORγt⁺IL-17A^hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Malt1A, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated DANKGTPEETGSYLVSK peptide (ubiquitinated K648 residue) of Malt1A. Parent ion corresponding to DANkGTPEETGSYLVSK peptide mass has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated. b Representative immunoblot of Flag and ubiquitin following polyubiquitinated protein enrichment of extracts using TUBE2 from CD90.1+ sorted EL4 cells transduced with MSCV-CD90.1-Flag-Malt1A or MSCV-CD90.1-Flag-Malt1A K648R retrovirus. c Flow cytometry analysis of intracellular IL-17A and intranuclear RORγt in CD90.1⁺ Malt1^−/− CD4⁺ T cells transduced with indicated retrovirus and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on CD90.1⁺ T cells. d MFI of Malt1A in CD90.1⁺ Malt1^−/−CD4⁺ T cells transduced with indicated retroviruses and in vitro polarized under Th17 condition. Data (n = 6) are mean of three independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t test. Source data are provided as a Source Data file