FIGURE 4.

Knockdown of TRAF5 decreases RORγt protein levels and ubiquitination of RORγt. A, FLAG-TRAF5 and shTRAF5-1, -2, and -3 were co-transfected into HEK293T cells. IB, immunoblot. B, naïve CD4⁺ T cells were polarized under Th17 conditions. Th17 cells were transduced with a lentivirus containing shCK, shTRAF5-1, or shTRAF5-2. The cells were treated with MG132 for 4 h before being harvested. Immunoprecipitation (IP) was carried out with anti-ubiquitin (Ubi) antibody. C, TRAF5 was knocked down in Th17 cells using shRNA lentivirus containing a puromycin resistance cassette. IL-17A, IL-17F, IL-21, IL-23R, RORγt, and TRAF5 mRNA levels of puromycin-selected cells were analyzed by quantitative RT-PCR. ns, not significant. Data represent at least three independent experiments, and error bars show mean ± S.D.