Ubiquitination of Stat3 at K180 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Stat3, 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 TLkSQGDMQDLNGNNQSVTR peptide (ubiquitinated K180 residue) of Stat3. Parent ion corresponding to TLkSQGDMQDLNGNNQSVTR peptide mass (774.0342, z = +3, retention time t = 38.1356 min) has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated and mass difference corresponding to GG tag (114.04293 Da) has been assigned to a K3 residue as indicated (K180-GG) by the difference between the y17 and y18 fragment ion masses. b Representative immunoblot of HA, Flag, Xpress, and GAPDH following Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, Flag-Stat3 K180R, and Xpress-Hectd3; data are representative of three independent experiments. c Flow cytometry analysis of intracellular IL-17A, and intranuclear RORγt and Stat3 in GFP + Stat3 −/− CD4 + T cells transduced with MSCV-Stat3 or MSCV-Stat3 K180R retroviruses and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on GFP + T cells. d MFI of pStat3 Y705 and Stat3 in GFP + Stat3 −/− CD4 + T cells transduced with indicated retroviruses normalized to those transduced with MSCV empty vector and in vitro polarized under Th17 conditions. Data ( n = 6) are mean of three (pStat3 Y705) and two (Stat3) 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
{}