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. 2014 Feb 19;9(2):e89479. doi: 10.1371/journal.pone.0089479

Figure 4. EVER2 deficiency impairs TRAF protein production.

Figure 4

(A–D) EV and Healthy cell lines were left untreated or stimulated with TNF for the time periods indicated. (A) Cytoplasmic extracts were subjected to western blotting. The results shown are representative of three independent experiments. (B) Whole-cell lysates were analyzed by western blotting after various times. (C) qRT-PCR was used to measure the induction of TRAF1 1.5–12 h after stimulation with TNF. Results are reported as fold-induction with respect to untreated cells. The basal level for the Healthy cell line was set to 1. A logarithmic (log) scale was used for the Y axis. Experiments were performed twice independently. (D) Nuclear extracts were subjected to immunoblot analysis with TRAF2 antibody for the times indicated. The results shown are representative of three independent experiments. (E–F) EV and Healthy (Heal.) cell lines were left untreated. (E) Cells were stained for TRAF2; the nucleus was stained with DAPI. Bars, 20 µm. (F) Keratinocytes were lysed in Triton X-100 lysis buffer. Soluble and insoluble fractions were subjected to immunoblot analysis with TRAF2 antibody. The TRAF2 and actin bands were quantified by densitometry for the soluble fraction. Results are reported as the ratio of TRAF2 to actin. The ratio obtained for the Healthy cell line was set to 1. (G) HEK-293T cells were transfected with plasmids encoding Flag-tagged EVER2 and Gluc2-tagged TRAF1, TRAF2 or RIPK1 proteins or Gluc2 fragment as a control (Gluc2-Ø). Crude lysates were immunoprecipitated with anti-Flag antibody (IP) and immunoblotted (IB) with the antibodies indicated. (H) Endogenous TRAF2 was immunoprecipitated (IP) with anti-TRAF2 in miEVER2 and control cells, and then subjected to immunoblotting (IB) with anti-Ubiquitin (Ub) antibody. The results shown are representative of three independent experiments. (I) HEK-293T cells were cotransfected with Gfp-tagged TRAF2, hemaglutinin (HA)-tagged Ubiquitin (Ub) with or without Flag-EVER2. After 24 h, the cells were treated with TNF for 2 h. Lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. The results shown are representative of two independent experiments.