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. 2018 Nov 5;215(11):2850–2867. doi: 10.1084/jem.20172026

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© 2018 Chen et al.

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Figure 9. — USP38 deubiquitinates TCR-induced K48-linked poly-ubiquitination of JunB. (A) Co-immunoprecipitation from lysates of HEK293T cells that were transfected with the indicated plasmids. Whole lysates were immunoprecipitated (IP) with anti-M2 and followed by immunoblotting (WB) with anti-HA and anti-M2. (B) Co-immunoprecipitation from lysates of CD4⁺ T cells that were treated with anti-CD3/28 for the indicated time points. Whole lysates were IP with anti-USP38 and followed by WB with the indicated antibodies. (C and D) Co-immunoprecipitation of proteins that were synthesized by in vitro translation system. The product mixture was IP with IgG (C) or anti-M2 (D) and followed by WB with anti-HA or anti-M2. (E and F) Immunoblot analyses of ubiquitinated JunB in HEK293T cells that were transfected with the indicated plasmids. Cell lysates were IP with anti-M2 and WB with anti-HA, anti-M2, or anti-USP38. K48, only Lys48 site left in ubiquitination. K63, only Lys63 site left in ubiquitination. K48LA, only Lys48 mutated into Ala in ubiquitination. USP38 mutants: 1M (C454S), 2M (C454S/H857A), and Cat (C454S/H857A/D918N). (G) Immunoblot analyses of USP38-mediated removal of JunB ubiquitination. Whole lysates of HEK293T cells transfected with the indicated plasmids were IP with anti-M2. The immunoprecipitates were then mixed with products from in vitro translation system containing WT USP38 or its DUB mutant (Cat) and followed by WB with the indicated antibodies. (H) Immunoblot analyses of ubiquitinated JunB in CD4⁺ T cells that were pretreated with or without MG132 and then treated with anti-CD3/28 for 2 h. Whole lysates were IP with anti-JunB and followed by WB with the indicated antibodies. (I) Immunoblot analyses of USP38-mediated removal of endogenous JunB ubiquitination. CD4⁺ T cells were pretreated with or without MG132 and stimulated with anti-CD3/CD28 for 2 h. Cell lysates were IP with anti-JunB. The immunoprecipitates were then mixed with WT mUSP38 or its DUB mutant from in vitro translation system and followed by WB with the indicated antibodies. Data are representative of three (A–D, F, and H) or two (E, G, and I) independent experiments.