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. 2019 Jul 25;10:1718. doi: 10.3389/fimmu.2019.01718

Figure 8.

Figure 8

PHA-767491 suppresses activity of transcription factors. (A) PHA-767491 impairs the activity of NFAT/AP-1. NFAT/AP-1-luciferase reporter Jurkat cells were treated with DMSO or PHA-767491 and stimulated with plate-bound CD3 antibodies for 6 h. (B) PHA-767491 impedes NF-κB activity. NF-κB-firefly luciferase and Renilla luciferase control plasmids were electroporated into Jurkat cells. The electroporated cells were cultured in hygromycin selection media before the assay. The NF-κB-firefly luciferase containing Jurkat cells were treated with DMSO or PHA-767491 and stimulated with plate-bound CD3 antibodies for 6 h. NF-κB activity is established as a ratio of the NF-κB-dependent firefly luciferase over the Renilla luciferase control. Data shown is representative of at least three independent experiments. Bar charts, represented as mean ± SEM, have been normalized to the NS sample. Statistical significance was determined by unpaired two-sided Student's t-test (**p < 0.01; ***p < 0.001). (C) Immunoblots of Jurkat cell lysates that were not-treated (–) or treated (+) with PHA-767491 and stimulated with plate-bound anti-CD3 antibodies for 9 h. Samples were either treated with zVAD-FMK at the indicated concentrations or not-treated with the caspase inhibitor. Representative blots of at least three independent experiments are shown.