Figure 4.

USP15 interacts with RORγt and deubiquitinates K446. (A) Control or RORγt-flag expressing EL-4 cells were established and intracellular IL-17 was detected by flow cytometry. IL-17-producing cells in gated area were enriched by IL-17 catching beads and analyzed for intracellular IL-17 (right panel). (B) Anti-flag antibody was used to IP lysates from control or RORγt-flag-expressing EL-4 cells and IP complexes were resolved on a protein gel. Arrows indicate the bands containing USP15 and RORγt on the silver stained gel. (C) RORγt-SRC1 interaction was detected by IB of anti-flag antibody IP cell lysates of control or RORγt-flag expressing EL-4 cells. (D) Naïve T cells were differentiated into Th17 cells for three days. Control IgG or αRORγt was used to perform IP to detect RORγt-USP15 interactions. HC indicates heavy chain. (E) WT USP15 or C298A was expressed with RORγt in 293T cells for 48 h. IP was performed to detect RORγt-USP15 interactions. Immunoprecipitated USP15 levels relative to that immunoprecipiated by wild type RORγt was calculated. HC indicates heavy chain. (F) Lysates of differentiated Th17 cells were incubated with or without Ub-HS-HA probe to covalently crosslink active deubiquitinases, which were then IP with αHA and resolved by SDS-PAGE. Arrow indicates the band containing USP15. (G) MS-identified USP15 peptides are highlighted by shadows. (H) RORγt was expressed alone or together with USP15 WT or inactive C298A mutant in the presence of HA-Ub in 293T cells for 48 h. RORγt was then IP and ubiquitinated RORγt (RORγt-Ub) was detected by IB with αHA. (I) K446 (all other Ks are mutated to Rs except K446) was expressed alone or together with USP15 WT or inactive C298A mutant in the presence of HA-Ub in 293T cells for 48 h. RORγt was then IP and ubiquitinated RORγt (RORγt-Ub) was detected by IB with αHA. Unless specified, data shown are the representative of at least three independent experiments.