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. 2015 Jul 28;290(36):22236–22249. doi: 10.1074/jbc.M115.653543

FIGURE 3.

FIGURE 3.

TRAF6 is demethylated and activated by JMJD6. A, Huh7.5 cells were treated with or without FSL-1 (TLR6/2 ligand) for 1 h, and TRAF6 was immunoprecipitated (IP) from cell lysates. Western blot analysis of JMJD6 and TRAF6 is shown. Immunoprecipitation with nonimmune IgG served as the negative control. B, immunoprecipitation with anti-methyl-arginine antibody and immunoblotting for TRAF6 from control cells or cells overexpressing JMJD6 is shown. MeR, proteins immunoprecipitated with anti-methyl-arginine antibody. C, in vitro self-ubiquitinylation of recombinant TRAF6 (0.2 μg per reaction) in the presence of 0.2 μg of JMJD6 with or without its required cofactor, α-ketoglutarate (α-K). Immunoblots are shown for ubiquitin-specific, methyl-arginine-specific, and TRAF6-specific antibodies. D, Huh7.5 cells were transfected with an HA-tagged modified ubiquitin containing Lys-63 as the only lysine with or without JMJD6, immunoprecipitated with anti-HA antibody, and probed for TRAF6 to detect Lys-63-linked poly-ubiquitinated TRAF6. IB, immunoblot. E, left panel, control Huh7.5 cells or cells expressing JMJD6 were co-transfected with wild type or dominant-negative (DN) TRAF6 and NF-κB luciferase reporter assay were performed. Right panel, RNA was isolated from control or JMJD6-transfected cells, and the indicated NF-κB target gene mRNA abundance was determined by RT-qPCR. Data are presented as the mean ± S.D. **, p < 0.01; *, p < 0.05. n = 3. ICAM, intercellular adhesion molecule 1. F, Western blot analysis of NF-κB p65 in nuclear fractions of control Huh7.5 cells and cells transfected with JMJD6. G, NF-κB luciferase reporter assay in THP-1 cells, either control cells (−) or cells expressing JMJD6 (J6) (left). Data are presented as the mean ± S.D. **, p < 0.01. n = 3. H, representative images of proximity ligation assays in THP-1 cells untreated or treated with LPS for 30 min (right). TRAF6 interaction with JMJD6 was detected using anti-JMJD6 and anti-TRAF6 antibodies. Negative control, signal in the absence of primary antibodies.