WASp is SUMO modified in vitro and in vivo. (A-B) In vitro SUMOylation using HeLa cells stably transfected with FLAG/Myc-tagged human full-length WASp construct. Immunoprecipitated WASp-FLAG incubated with SUMO-1, -2, and -3 (S1, S2, S3) individually or with the indicated combinations of SUMO-proteins sequentially probed with SUMO-1, -2/3, and WASp Abs in western blot assays (A). Purified RanGAP1 (RG1) protein, provided with the assay kit, served as a positive control and was coincubated with all 3 SUMO proteins (S1, S2, S3) together. RG1(+), RanGap1, E1, and E2, ATP included (+); RG(−), RanGap1, E1, E2 included but ATP excluded from the assay. WASp-FLAG samples incubated with SUMO1 (S1) or with the SUMO1 mutant lacking enzymatic activity (S1-mutated [mu]) (B). The data are representative of 3 biologic replicates for A and 2 biologic replicates for B. Numbers indicate the molecular masses (kDa) of key bands. SUMOylated-WASp is indicated as WASp*SUMO conjugates at ∼85 kDa and above. NEM−, assay performed in the absence of NEM, a chemical that inhibits de-SUMOylation. (C) In vivo SUMOylation. Nuclear (nu) and cytoplasmic (cy) extracts of NEM-treated, T_H1- or T_H17-skewed/TCR-activated or T_H0-nonskewed/TCR-nonactivated primary T_H-cells in the presence (+) or absence (−) of NEM sequentially reprobed with the indicated antibodies by western blot. (D) RNA interference-mediated depletion of endogenous SUMO1 was performed in primary T_H1-skewed/TCR-activated cells using SUMO1-shRNA or scrambled-shRNA at day 6 after transfection and NEM treatment (supplemental Figure 2B for temporal progression of SUMO1 k/d) and western blots sequentially reprobed with the indicated Abs. (supplemental Table 1 for reagents). Data are representative of 2 independent experiments. NEM^+/−, treated/nontreated with NEM.