Extended Data Fig. 4: Usp22 acts as a deubiquitinase to control post-translational Foxp3 expression.
From: CRISPR screen in regulatory T cells reveals modulators of Foxp3
a, Endogenous interaction of Usp22 and Foxp3 in mouse iTreg cells from WT mice. Rabbit Usp22 antibody was used to perform the immunoprecipitation and mouse Foxp3 antibody was used to detect the bound Foxp3. Normal rabbit IgG was used as control. Whole-cell lysates (WCL) were used as sample processing controls. b, Ubiquitination assay of Foxp3. HEK293 cells were cotransfected with Flag–Foxp3 and HA–ubiquitin (HA–ub) and either Myc-empty vector, Myc–Usp22, or the catalytically inactive mutant Myc–Usp22(C185A) (C>A), and then immunoprecipitated with anti-Flag and immunoblotted for HA-ubiquitin (Foxp3-ub). Whole-cell lysates were used as sample processing controls. c, Splenocytes isolated from Usp22+/+Foxp3YFP-cre WT or Usp22fl/flFoxp3YFP-cre KO mice were treated with 200 μg ml−1 cycloheximide for the indicated times. Inset numbers for each histogram indicate the MFI of Foxp3 in Treg cells (black, WT; blue, KO). d, Foxp3 MFI from splenic CD4+CD25+Foxp3+ Treg population treated with 200 μg ml−1 cycloheximide for the indicated time course (n = 3), corresponding to c. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in Methods, ‘Statistics and reproducibility’. Source data for blots can be found in Supplementary Fig. 1.
