(A–C) IP analysis of the RORγt-CBFβ interaction in HEK293T cells with indicated expression plasmids for WT (A), deletion mutation (B), or alanine scanning mutation (C) RORγt. ΔDBD, DNA binding domain-deleted RORγt; ΔHinge, hinge-deleted RORγt; ΔLBD, Lihand-binding domain-deleted RORγt. (D) The percentage of Th17 differentiation activity supported by the RORγt with the indicated point mutation expressed in RORγt^–/– CD4⁺ T cells relative to those expressing WT RORγt (defined as 100%). RORγt^–/– CD4⁺ T cells expressing GFP alone (EV) or with WT RORγt or RORγt with the indicated single amino acid mutated to alanine were polarized under Th17 conditions. (E) Visualization of computer-predicted interaction between RORγt and CBFβ. The cyan fragment indicates RORγt amino acids 403–413. R407, L410, and E412 of RORγt critical for binding to CBFβ are indicated. Red dashed lines indicate the ionic lock between R407 and E336. (F) IP analysis of CBFγ interaction with RORβt or R407A/L410A DM. (G and H) Relative luciferase activity from HEK293T cells transfected with pGL3 control or RORγt reporter (RORBE) together with plasmids expressing WT or RORγt with indicated mutation (G) with or without CBFβ (H). (I) The percentage of Th17 differentiation activity supported by the RORγt with indicated mutation expressed in RORγt^–/– CD4⁺ T cells relative to those expressing WT RORγt (defined as 100%). (J) Flow cytometric analysis of CD4⁺ and CD8⁺ thymocytes ex vivo developed for 3 days from sorted RORγt^–/– CD4^–CD8^– transduced with WT or RORγt with indicated mutation. Right: The summary of the percentages of CD4⁺ plus CD4⁺CD8⁺ T cells among live Thy1.2⁺ cells. Data represent 3 (D and G) or 4 (H–J) independent experiments; representative Western blots are shown from 3 independent experiments (A–C, and F). Data were analyzed by 1-way ANOVA with Dunnett’s (D) or Tukey’s (G–J) post hoc test. *P < 0.05; **P < 0.01. WCL, whole-cell lysate.