Figure 3.
ETS1 and NF-κB p65 are the key transcriptional factors regulating IL27Ra expression. (A) The transcriptional activity analysis of the truncated constructs in the IL27Ra promoter region by luciferase assay (see Methods). Data are shown as mean ± SEM. (B-C) Histograms showing the transcriptional activity of IL27Ra upon overexpression of (B) NF-κB p65 and (C) ETS1 by luciferase assay. Data are shown as mean ± SEM. (D) This histogram displays the IL27Ra promoter-mutation (the sequence GGGA was mutated to TTTT within the predicted NF-kB p65 binding sites) abolishes the NF-κB p65 induced IL27Ra transcription by luciferase assay (supplemental Figure 3D; supplemental Methods). Data are shown as mean ± SEM. (E) The IL27Ra promoter-mutation (the sequence CCCTGAAGG was mutated to AAAAAGGTT within the predicted ETS1 binding sites) abolishes the ETS1 induced IL27Ra transcription by luciferase assay (supplemental Figure 3D; supplemental Methods). Data are shown as mean ± SEM. (F-G) Freshly isolated KSL cells were infected by lentivirus carrying ETS1 shRNA or scramble control, and 3 days later, 105 GFP+ cells were purified for western blot and quantitative PCR to evaluate the expression of ETS1 and IL27Ra. (F) Representative western blot showing the expression of ETS1 and IL27Ra, (G) This histogram depicts the expression of ETS1 and IL27Ra. Data are shown as mean ± SEM. (H) This histogram displays the expression of IL27Ra in KSL cells in response to IKK inhibitor (2 μM TPCA-1, 8 days) treatment. Data are shown as mean ± SEM. (I-K) Freshly isolated KSL cells were infected by lentivirus overexpressing ETS1 or NF-κB p65. GFP+ cells were purified 4 days after infection, and were subjected to reverse transcriptase-PCR. (I) Experimental design to evaluate the effect of overexpressed ETS1 or NF-κB p65 on IL27Ra. (J) This histogram depicts the messenger RNA expression of Ets1 and IL27Ra in response to ETS1 overexpression, (K) This histogram depicts the messenger RNA expression of NF-κB p65 and IL27Ra in response to NF-κB p65 overexpression. Data are shown as mean ± SEM.

ETS1 and NF-κB p65 are the key transcriptional factors regulating IL27Ra expression. (A) The transcriptional activity analysis of the truncated constructs in the IL27Ra promoter region by luciferase assay (see Methods). Data are shown as mean ± SEM. (B-C) Histograms showing the transcriptional activity of IL27Ra upon overexpression of (B) NF-κB p65 and (C) ETS1 by luciferase assay. Data are shown as mean ± SEM. (D) This histogram displays the IL27Ra promoter-mutation (the sequence GGGA was mutated to TTTT within the predicted NF-kB p65 binding sites) abolishes the NF-κB p65 induced IL27Ra transcription by luciferase assay (supplemental Figure 3D; supplemental Methods). Data are shown as mean ± SEM. (E) The IL27Ra promoter-mutation (the sequence CCCTGAAGG was mutated to AAAAAGGTT within the predicted ETS1 binding sites) abolishes the ETS1 induced IL27Ra transcription by luciferase assay (supplemental Figure 3D; supplemental Methods). Data are shown as mean ± SEM. (F-G) Freshly isolated KSL cells were infected by lentivirus carrying ETS1 shRNA or scramble control, and 3 days later, 105 GFP+ cells were purified for western blot and quantitative PCR to evaluate the expression of ETS1 and IL27Ra. (F) Representative western blot showing the expression of ETS1 and IL27Ra, (G) This histogram depicts the expression of ETS1 and IL27Ra. Data are shown as mean ± SEM. (H) This histogram displays the expression of IL27Ra in KSL cells in response to IKK inhibitor (2 μM TPCA-1, 8 days) treatment. Data are shown as mean ± SEM. (I-K) Freshly isolated KSL cells were infected by lentivirus overexpressing ETS1 or NF-κB p65. GFP+ cells were purified 4 days after infection, and were subjected to reverse transcriptase-PCR. (I) Experimental design to evaluate the effect of overexpressed ETS1 or NF-κB p65 on IL27Ra. (J) This histogram depicts the messenger RNA expression of Ets1 and IL27Ra in response to ETS1 overexpression, (K) This histogram depicts the messenger RNA expression of NF-κB p65 and IL27Ra in response to NF-κB p65 overexpression. Data are shown as mean ± SEM.

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