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. 2018 Mar 26;293(19):7268–7280. doi: 10.1074/jbc.RA117.001684

Figure 3.

Figure 3.

The significance of the phosphorylation event in TIFA-mediated NF-κB activation. a, the vectors expressing full-length TIFA, the FHA domain (TIFA FHA), the R51A/K88A/N89A (MT1), or the G50E/S66A (MT2) mutants of full-length TIFA were transfected in HeLa cells with NT or treatment of ETO. The mRNA levels of the indicated genes were examined using quantitative RT-PCR analysis. Data were represented as the mean ± S.D. from eight independent experiments. **, p < 0.01 (Student's t test). b, the lysates of TIFA stably expressed HeLa cells with NT or treatment of ETO were subjected to immunoprecipitation using FLAG antibody and probed with the indicated antibodies. Before harvesting cells, cells were treated with formaldehyde for 10 min. c, FLAG-TIFA was co-transfected with WT H2AX (WT), nonphosphorylatable mutant S139A of H2AX (SA), or phosphorylation mimicking mutant S139E of H2AX (SE) in HeLa cells. Cells described were treated with ETO or LPS and the mRNA levels of the indicated genes were examined using quantitative RT-PCR analysis. Data were represented as the mean ± S.D. from six independent experiments. **, p < 0.01 (Student's t test). d, whole cell lysates and chromatin fractions from HeLa cells expressing TIFA or T9A mutant upon damage treatment were subjected to Western blot analysis probed with the indicated antibodies. e, whole cell lysates from HeLa cells expressing vector, TIFA, and T9A mutant upon damage treatment were subjected to Western blot analysis probed with indicated antibodies. f, the total mRNA was prepared from cells described in e and the mRNA levels of the indicated genes were examined using quantitative RT-PCR analysis. Data were represented as the mean ± S.D. from three independent experiments. **, p < 0.01 (Student's t test). g, cells were transfected with FLAG-tagged TIFA or T9A mutant as indicated with or without DNA damage treatment. Cellular extracts were prepared and immunoprecipitation was performed with FLAG antibody. IgG light chain is indicated with *. IP, immunoprecipitation; n.s., nonspecific.