And Hamilton 1995). To investigate how the IFN– and NF-B-ip-10 INDUCTION BY IFN- AProbe (ISRE) Antibody Competitor IFN wt ??wt ??+ m wt wt ?IRF1 p65 ???+ + + wt wt wt wt wt ???Stat1 Stat1 ?wt m GAS ?+ + + + +821 BIRF1 wt IFN- (4 h) ?+ ?IRF1 null +irfip-10 1 2 3 4 5 6 7 8 9 10 gapdhCIFN- (4 h) 4-OH-tamoxifen (4 h) ip-10 ??IRF1 null ?+ + ?+ + ??IRF1 null + IRF1-ER ?+ + ?+ +gapdhDIFN- (h) IFN- (h) ??0.5 ?IP: anti-IRF1 2 ??0.EIFN- (h) ip-10 ?IP: anti-IRF1 IKK??2 IKK+/+ ?2 ip-10 ip-NCINPUTip-INPUTFIG. 3. IRF1 recruitment to the ip-10 promoter by interferon (IFN)- requires IKK-. (A) Wild-type MEFs were untreated s11606-015-3271-0 (lane 1) or treated for 4 h with 1,000 IU/mL of murine IFN- (lanes 2?0) and nuclear lysates were analyzed by electrophoretic mobility shift assay (EMSA) using the IFN-stimulated regulatory element (ISRE) from the ip-10 promoter. A mutant version of the ISRE probe did not produce a band shift (lane 3). In addition, adding anti-IRF1 (lane 4) supershifted the ISREbound complex, while anti-p65 did not (lane 5), nor did 2 different antibodies to Stat1 (lanes 9 and 10). Finally, competition with a 50-fold excess of unlabeled wild-type ISRE probe competed the labeled probe while a 50-fold excess of mutant ISRE and wild-type GAS probe did not (lanes 6, 7, and 8, respectively). (B) IRF1-null MEFs and wild-type MEFs derived from littermates were treated for 4 h with IFN- and RNA was analyzed by the Northern method for the expression of ip-10, irf1, and gapdh mRNAs. (C) IRF1-null cells or the same cells in which IRF1 expression was stably restored with a retrovirus expressing an estrogen receptor-IRF1 fusion protein were treated with 1 M tamoxifen, IFN-, or both, and ip-10 and gapdh mRNA expression were measured by the Northern method. (D) Wild-type MEFs or (E) IKK–null cells or a pool of the same cells in which IKK- expression was stably restored were treated with either IFN- or IL-1 and IRF1 binding to the ip-10 promoter was analyzed by ChIP. The lane at the far right of (D) represents a control in which the lysate was immunoprecipitated with an isotype control antibody.result indicates strongly that the additional BQ-123 clinical trials component BQ-123 web required for IFN–induced ip-10 transcription shown in Figure 3C, as well as the signal provided by IL-1 for synergy with IFN-, is not classical NF-B.DiscussionIFN- binds to its receptor, followed by the sequential activation of Jaks 2 and 1, which in turn phosphorylate the cytoplasmic tail of the receptor at conserved tyrosine residues.One site in particular is essential for docking Stat1 (Qing journal.pone.0158910 and others 2005). Following binding, Stat1 is itself phosphorylated by the Jaks, causing it to homodimerize and translocate to the nucleus, where it binds to GAS elements in the promoters of ISGs. By this mechanism, IFN- induces sustained IRF1 expression, which participates in the transcription of a subset of ISGs, represented here by ip-10, by binding to their ISREs. It is clear from the work shown here that other IFN- signals are required in order for ip-10 to be induced. We have shown previously that irf1 expression does not require822 AIFN- (h) ?IL-1 (h) ??1 ?2 ?4 1 ?2 ?4 ?1 1 2 2 4SHULTZ ET AL. BIFN- (h) IL-1 (h) ??1 ??1 1 1 2 ??2 2 2 4 ??4 4ip-irfgapdhCIFN- (h) IL-1 (h) ??0.25 0.5 ??1 ?2 ????1 ?2 0.25 0.5 0.25 0.5 1 1 2 2 0.25 0.Bp65:p65 p65:pBFIG. 4. Interferon (IFN)- and interleukin (IL)-1 induce ip-10 synergistically. (A) Wild-type MEFs were treated with murine IL-1 (10 mg/mL) or IFN- (1,000 IU/mL) and RNA was analyzed b.And Hamilton 1995). To investigate how the IFN– and NF-B-ip-10 INDUCTION BY IFN- AProbe (ISRE) Antibody Competitor IFN wt ??wt ??+ m wt wt ?IRF1 p65 ???+ + + wt wt wt wt wt ???Stat1 Stat1 ?wt m GAS ?+ + + + +821 BIRF1 wt IFN- (4 h) ?+ ?IRF1 null +irfip-10 1 2 3 4 5 6 7 8 9 10 gapdhCIFN- (4 h) 4-OH-tamoxifen (4 h) ip-10 ??IRF1 null ?+ + ?+ + ??IRF1 null + IRF1-ER ?+ + ?+ +gapdhDIFN- (h) IFN- (h) ??0.5 ?IP: anti-IRF1 2 ??0.EIFN- (h) ip-10 ?IP: anti-IRF1 IKK??2 IKK+/+ ?2 ip-10 ip-NCINPUTip-INPUTFIG. 3. IRF1 recruitment to the ip-10 promoter by interferon (IFN)- requires IKK-. (A) Wild-type MEFs were untreated s11606-015-3271-0 (lane 1) or treated for 4 h with 1,000 IU/mL of murine IFN- (lanes 2?0) and nuclear lysates were analyzed by electrophoretic mobility shift assay (EMSA) using the IFN-stimulated regulatory element (ISRE) from the ip-10 promoter. A mutant version of the ISRE probe did not produce a band shift (lane 3). In addition, adding anti-IRF1 (lane 4) supershifted the ISREbound complex, while anti-p65 did not (lane 5), nor did 2 different antibodies to Stat1 (lanes 9 and 10). Finally, competition with a 50-fold excess of unlabeled wild-type ISRE probe competed the labeled probe while a 50-fold excess of mutant ISRE and wild-type GAS probe did not (lanes 6, 7, and 8, respectively). (B) IRF1-null MEFs and wild-type MEFs derived from littermates were treated for 4 h with IFN- and RNA was analyzed by the Northern method for the expression of ip-10, irf1, and gapdh mRNAs. (C) IRF1-null cells or the same cells in which IRF1 expression was stably restored with a retrovirus expressing an estrogen receptor-IRF1 fusion protein were treated with 1 M tamoxifen, IFN-, or both, and ip-10 and gapdh mRNA expression were measured by the Northern method. (D) Wild-type MEFs or (E) IKK–null cells or a pool of the same cells in which IKK- expression was stably restored were treated with either IFN- or IL-1 and IRF1 binding to the ip-10 promoter was analyzed by ChIP. The lane at the far right of (D) represents a control in which the lysate was immunoprecipitated with an isotype control antibody.result indicates strongly that the additional component required for IFN–induced ip-10 transcription shown in Figure 3C, as well as the signal provided by IL-1 for synergy with IFN-, is not classical NF-B.DiscussionIFN- binds to its receptor, followed by the sequential activation of Jaks 2 and 1, which in turn phosphorylate the cytoplasmic tail of the receptor at conserved tyrosine residues.One site in particular is essential for docking Stat1 (Qing journal.pone.0158910 and others 2005). Following binding, Stat1 is itself phosphorylated by the Jaks, causing it to homodimerize and translocate to the nucleus, where it binds to GAS elements in the promoters of ISGs. By this mechanism, IFN- induces sustained IRF1 expression, which participates in the transcription of a subset of ISGs, represented here by ip-10, by binding to their ISREs. It is clear from the work shown here that other IFN- signals are required in order for ip-10 to be induced. We have shown previously that irf1 expression does not require822 AIFN- (h) ?IL-1 (h) ??1 ?2 ?4 1 ?2 ?4 ?1 1 2 2 4SHULTZ ET AL. BIFN- (h) IL-1 (h) ??1 ??1 1 1 2 ??2 2 2 4 ??4 4ip-irfgapdhCIFN- (h) IL-1 (h) ??0.25 0.5 ??1 ?2 ????1 ?2 0.25 0.5 0.25 0.5 1 1 2 2 0.25 0.Bp65:p65 p65:pBFIG. 4. Interferon (IFN)- and interleukin (IL)-1 induce ip-10 synergistically. (A) Wild-type MEFs were treated with murine IL-1 (10 mg/mL) or IFN- (1,000 IU/mL) and RNA was analyzed b.
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