Fig. 3
From: K27-linked RORγt ubiquitination by Nedd4 potentiates Th17-mediated autoimmunity
RORγt PPLY Motif Binds to the WW Domains of Nedd4. A Identification of a conserved Nedd4-binding motif in RORγt ligand-binding domain. Schematic diagram of the domain structure of RORs. Conserved sequence (PPLYKEL) of the AF2 domain among members of the ROR family (upper). Schematic diagram of the domain structure of Nedd4 (lower). B Immunoblot analysis of RORγt immunopreciptates of lysates of CD4+ T cells stimulated under Th17-biased condition (anti-CD3, anti-CD28, TGF-β, and IL-6) for three days, and restimulated with anti-CD3 and anti-CD28, with antibodies against Nedd4 and Nedd4-2. The relative expression of Nedd4 and Nedd4-2 to RORγt were shown under the WB bands. **p < 0.01; student t test. C Immunoblot analysis of Myc immunoprecipitates of HEK293T cells transfected with Flag-tagged Nedd4, Nedd4 WW domains, Nedd4 C2 domain, Nedd4∆C2, or Nedd4∆WW, together with Myc-tagged RORγt, with anti-Flag. D GST-Nedd4 and GST-Nedd4 WW domain pull-down assay using Th17 cell lysates. E Immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4, and Myc-tagged RORγt or RORγt Y479F mutant, with anti-Flag. F EAE scores of Tcrb–/– mice (n = 7 mice per group) receiving CD4+ T cells from WT and RorcY479F mice immunized with immunization with MOG35-55 in CFA. ***p < 0.001; Mann-Whitney U test. G Flow cytometric analysis of Th1/Th17 cells in the dLNs of Tcrb–/– mice (n = 3 mice per group) received CD4+ T cells (5 × 106) from WT and RorcY479F mice at day 8 after immunization. ***p < 0.001; student t test. Data are representative of three independent experiments for B-E, and two independent experiments for F and G