Figure 3
Figure 3. Functional consequences of TNFAIP3 mutations. (A) Dose-dependent inhibitory effect of wild-type (WT) and mutant A20 on TNFα-induced NF-kB activation. Increasing amounts of plasmids encoding either WT or mutant A20 (rs2230926G) were transfected into human embryonic kidney (HEK) 293T cells, together with the NFκB-luciferase reporter and plasmid micro RNA β-galactosidase plasmids. The following day, cells were stimulated for 6 hours with 10 ng/mL TNFα. Luciferase activity was normalized to β-galactosidase activity, and ratios were expressed as percentage of NF-kB activation compared with that of the control without A20 plasmid, which was set at 100%. Results are means ± standard error of the mean of 6 determinations. (B) Inhibitory effect of WT and mutant A20 on TNFα-induced NF-kB activation. Transfection assays were performed as described earlier. Results are means ± standard error of the mean of several independent experiments performed with 6 to 35 determinations: 8 experiments for the WT A20 (n = 103 determinations), 7 experiments for the rs2230926G mutant (n = 101), and 3 experiments for the InsGG mutant (New 3; n = 44). (C) Western blot analysis of A20 protein variants. Total protein extracts were prepared from the same transfected-HEK 293T cells as described earlier, and western blot analysis of A20 protein variants demonstrated that the WT and the rs2230926 variant A20 proteins were expressed at the same level in our transfection assay. Of note, A20 monoclonal antibody, which recognizes an epitope located on exon 7, cannot detect the InsGG A20 protein variant that lacks the site of recognition of the antibody. However, the InsGG protein variant was expressed in transfected cells because it is able to partly inhibit NF-kB activation in response to TNF activation. (D) Immunodetection of A20 proteins in HEK 293T cells: HEK 293T cells were transiently transfected with the expression vector encoding A20 (WT, rs2230926G, or InsGG). Sixteen hours after transfection, cells were stimulated with TNF (10 ng/mL) for 6 hours before fixation. Cells were immunolabeled with anti-FLAG (M2 produced in mouse; Sigma). Nuclei were 4′,6-diamidino-2-phenylindole stained. ×40 magnification.

Functional consequences of TNFAIP3 mutations. (A) Dose-dependent inhibitory effect of wild-type (WT) and mutant A20 on TNFα-induced NF-kB activation. Increasing amounts of plasmids encoding either WT or mutant A20 (rs2230926G) were transfected into human embryonic kidney (HEK) 293T cells, together with the NFκB-luciferase reporter and plasmid micro RNA β-galactosidase plasmids. The following day, cells were stimulated for 6 hours with 10 ng/mL TNFα. Luciferase activity was normalized to β-galactosidase activity, and ratios were expressed as percentage of NF-kB activation compared with that of the control without A20 plasmid, which was set at 100%. Results are means ± standard error of the mean of 6 determinations. (B) Inhibitory effect of WT and mutant A20 on TNFα-induced NF-kB activation. Transfection assays were performed as described earlier. Results are means ± standard error of the mean of several independent experiments performed with 6 to 35 determinations: 8 experiments for the WT A20 (n = 103 determinations), 7 experiments for the rs2230926G mutant (n = 101), and 3 experiments for the InsGG mutant (New 3; n = 44). (C) Western blot analysis of A20 protein variants. Total protein extracts were prepared from the same transfected-HEK 293T cells as described earlier, and western blot analysis of A20 protein variants demonstrated that the WT and the rs2230926 variant A20 proteins were expressed at the same level in our transfection assay. Of note, A20 monoclonal antibody, which recognizes an epitope located on exon 7, cannot detect the InsGG A20 protein variant that lacks the site of recognition of the antibody. However, the InsGG protein variant was expressed in transfected cells because it is able to partly inhibit NF-kB activation in response to TNF activation. (D) Immunodetection of A20 proteins in HEK 293T cells: HEK 293T cells were transiently transfected with the expression vector encoding A20 (WT, rs2230926G, or InsGG). Sixteen hours after transfection, cells were stimulated with TNF (10 ng/mL) for 6 hours before fixation. Cells were immunolabeled with anti-FLAG (M2 produced in mouse; Sigma). Nuclei were 4′,6-diamidino-2-phenylindole stained. ×40 magnification.

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