T difficult to obtainIRF1 binds to the ip-10 ISREIRF1 journal.pone.0111391 is an

T difficult to obtainIRF1 binds to the ip-10 ISREIRF1 is an ISRE-binding protein whose synthesis is induced in response to IFN- (Taniguchi and others 2001). We tested its binding to the ISRE in the ip-10 promoter by supershift analysis (Fig. 3A). Anti-IRF1 get JC-1 supershifted the j.addbeh.2012.10.012 ISRE-binding complex formed in response to a 4-h treatment with IFN- (lane 4). Anti-p65 (an isotype control), on the other hand, did not (lane 5), as expected since p65 binds to B elements, which were not present in the probe. Furthermore, a version of the ISRE probe carrying mutations in the core ISRE-binding domain (GGAAAGTGAAACC GGACAGTGACACC) did not bind at all (lane 3). In the same gel, we also ran samples from cells treated with IFN- for 4 h and then preincubated with a 50-fold excess of Pyrvinium pamoate cost unlabeled order Pyrvinium embonate Wild-type ISRE probe, mutant ISRE probe, or GAS probe (lanes 6, 7, and 8, respectively). Only the unlabeled wild-type ISRE probe competed successfully, showing that the complex is specific for that sequence. Finally, two different antibodies that shift Stat1 bound to a GAS probe (data not shown) did not shift the ISRE-bound complex (lanes 9 and 10). These results led us to investigate whether IRF1-null MEFs induce ip-10 in response to IFN-. As shown in Figure 3B, these cells expressed much less ip-10 in response to IFN- after 4 h compared to match wild-type MEFs. The fact that a little IP-10 protein is still induced in these IRF1-null cells suggests partial compensation, perhaps through different IRFs. To determine if IRF1 expression could restore ip-10 induction to wild-type levels, we created an expression construct encoding a variant of the estrogen receptor (ER) fused to IRF1. The rationale is that constitutively expressed IRF1 translocates to the nucleus, causing cellular senescence. On the other hand, the nuclear translocation of IRF1-ER is tightly regulated and, in the absence of estrogen (or an estrogen mimic), it remains in the cytoplasm (Kroger and others 2003). IRF1-null cells expressed very little ip-10 mRNA in response to IFN-, either alone or in combination with tamoxifen (Fig. 3C). Interestingly, when IRF1-null cells stably expressing the IRF1-ER protein were treated with tamoxifenAIFN- (h) -IP: anti-p65 0.5 2 ip-10 ip-INPUTBIP: anti-p65 IKK??IKK+/+ 2 2 ip-IFN- (h)-INPUTip-FIG. 1. Interferon (IFN)- stimulates p65 binding to the ip-10 promoter in an IKK–dependent fashion. (A) Wildtype MEFs or (B) IKK–null MEFs or a pool of the same cells in which IKK- was stably restored were treated with 1,000 IU/mL of get Vasoactive Intestinal Peptide (human, rat, mouse, rabbit, canine, porcine) murine IFN-. Cross-linked chromatin fragments were immunoprecipitated with anti-p65. The recovered DNA was amplified by PCR with 32P-labeled -dCTP, using primers specific for the promoter region of ip-10. The products were separated in an 8 polyacrylamide gel, dried, and analyzed by autoradiography. An example representative of three experiments is shown. The lane at the far left of B represents a sample immunoprecipitated with an isotypematched control antibody.820 A BFold-Change ip-10 Primary RNA Relative to gapdhSHULTZ ET AL.IFN- (h) ip-gapdhIFN- (h)CIFN- (h) 0 0.75 1 2FIG. 2. Ip-10 is transcribed with delayed kinetics due to synthesis of an interferon (IFN)-stimulated regulatory element (ISRE)-binding factor. (A) Wild-type MEFs were treated with 1,000 IU/mL of murine IFN- for various times and ip-10 and gapdh mRNAs were measured by the Northern method. (B) The rate of ip-10 transcription in wild-type MEFs in response to IFN- measured by qPCR, using.T difficult to obtainIRF1 binds to the ip-10 ISREIRF1 is an ISRE-binding protein whose synthesis is induced in response to IFN- (Taniguchi and others 2001). We tested its binding to the ISRE in the ip-10 promoter by supershift analysis (Fig. 3A). Anti-IRF1 supershifted the j.addbeh.2012.10.012 ISRE-binding complex formed in response to a 4-h treatment with IFN- (lane 4). Anti-p65 (an isotype control), on the other hand, did not (lane 5), as expected since p65 binds to B elements, which were not present in the probe. Furthermore, a version of the ISRE probe carrying mutations in the core ISRE-binding domain (GGAAAGTGAAACC GGACAGTGACACC) did not bind at all (lane 3). In the same gel, we also ran samples from cells treated with IFN- for 4 h and then preincubated with a 50-fold excess of unlabeled wild-type ISRE probe, mutant ISRE probe, or GAS probe (lanes 6, 7, and 8, respectively). Only the unlabeled wild-type ISRE probe competed successfully, showing that the complex is specific for that sequence. Finally, two different antibodies that shift Stat1 bound to a GAS probe (data not shown) did not shift the ISRE-bound complex (lanes 9 and 10). These results led us to investigate whether IRF1-null MEFs induce ip-10 in response to IFN-. As shown in Figure 3B, these cells expressed much less ip-10 in response to IFN- after 4 h compared to match wild-type MEFs. The fact that a little IP-10 protein is still induced in these IRF1-null cells suggests partial compensation, perhaps through different IRFs. To determine if IRF1 expression could restore ip-10 induction to wild-type levels, we created an expression construct encoding a variant of the estrogen receptor (ER) fused to IRF1. The rationale is that constitutively expressed IRF1 translocates to the nucleus, causing cellular senescence. On the other hand, the nuclear translocation of IRF1-ER is tightly regulated and, in the absence of estrogen (or an estrogen mimic), it remains in the cytoplasm (Kroger and others 2003). IRF1-null cells expressed very little ip-10 mRNA in response to IFN-, either alone or in combination with tamoxifen (Fig. 3C). Interestingly, when IRF1-null cells stably expressing the IRF1-ER protein were treated with tamoxifenAIFN- (h) -IP: anti-p65 0.5 2 ip-10 ip-INPUTBIP: anti-p65 IKK??IKK+/+ 2 2 ip-IFN- (h)-INPUTip-FIG. 1. Interferon (IFN)- stimulates p65 binding to the ip-10 promoter in an IKK–dependent fashion. (A) Wildtype MEFs or (B) IKK–null MEFs or a pool of the same cells in which IKK- was stably restored were treated with 1,000 IU/mL of murine IFN-. Cross-linked chromatin fragments were immunoprecipitated with anti-p65. The recovered DNA was amplified by PCR with 32P-labeled -dCTP, using primers specific for the promoter region of ip-10. The products were separated in an 8 polyacrylamide gel, dried, and analyzed by autoradiography. An example representative of three experiments is shown. The lane at the far left of B represents a sample immunoprecipitated with an isotypematched control antibody.820 A BFold-Change ip-10 Primary RNA Relative to gapdhSHULTZ ET AL.IFN- (h) ip-gapdhIFN- (h)CIFN- (h) 0 0.75 1 2FIG. 2. Ip-10 is transcribed with delayed kinetics due to synthesis of an interferon (IFN)-stimulated regulatory element (ISRE)-binding factor. (A) Wild-type MEFs were treated with 1,000 IU/mL of murine IFN- for various times and ip-10 and gapdh mRNAs were measured by the Northern method. (B) The rate of ip-10 transcription in wild-type MEFs in response to IFN- measured by qPCR, using.T difficult to obtainIRF1 binds to the ip-10 ISREIRF1 is an ISRE-binding protein whose synthesis is induced in response to IFN- (Taniguchi and others 2001). We tested its binding to the ISRE in the ip-10 promoter by supershift analysis (Fig. 3A). Anti-IRF1 supershifted the j.addbeh.2012.10.012 ISRE-binding complex formed in response to a 4-h treatment with IFN- (lane 4). Anti-p65 (an isotype control), on the other hand, did not (lane 5), as expected since p65 binds to B elements, which were not present in the probe. Furthermore, a version of the ISRE probe carrying mutations in the core ISRE-binding domain (GGAAAGTGAAACC GGACAGTGACACC) did not bind at all (lane 3). In the same gel, we also ran samples from cells treated with IFN- for 4 h and then preincubated with a 50-fold excess of unlabeled wild-type ISRE probe, mutant ISRE probe, or GAS probe (lanes 6, 7, and 8, respectively). Only the unlabeled wild-type ISRE probe competed successfully, showing that the complex is specific for that sequence. Finally, two different antibodies that shift Stat1 bound to a GAS probe (data not shown) did not shift the ISRE-bound complex (lanes 9 and 10). These results led us to investigate whether IRF1-null MEFs induce ip-10 in response to IFN-. As shown in Figure 3B, these cells expressed much less ip-10 in response to IFN- after 4 h compared to match wild-type MEFs. The fact that a little IP-10 protein is still induced in these IRF1-null cells suggests partial compensation, perhaps through different IRFs. To determine if IRF1 expression could restore ip-10 induction to wild-type levels, we created an expression construct encoding a variant of the estrogen receptor (ER) fused to IRF1. The rationale is that constitutively expressed IRF1 translocates to the nucleus, causing cellular senescence. On the other hand, the nuclear translocation of IRF1-ER is tightly regulated and, in the absence of estrogen (or an estrogen mimic), it remains in the cytoplasm (Kroger and others 2003). IRF1-null cells expressed very little ip-10 mRNA in response to IFN-, either alone or in combination with tamoxifen (Fig. 3C). Interestingly, when IRF1-null cells stably expressing the IRF1-ER protein were treated with tamoxifenAIFN- (h) -IP: anti-p65 0.5 2 ip-10 ip-INPUTBIP: anti-p65 IKK??IKK+/+ 2 2 ip-IFN- (h)-INPUTip-FIG. 1. Interferon (IFN)- stimulates p65 binding to the ip-10 promoter in an IKK–dependent fashion. (A) Wildtype MEFs or (B) IKK–null MEFs or a pool of the same cells in which IKK- was stably restored were treated with 1,000 IU/mL of murine IFN-. Cross-linked chromatin fragments were immunoprecipitated with anti-p65. The recovered DNA was amplified by PCR with 32P-labeled -dCTP, using primers specific for the promoter region of ip-10. The products were separated in an 8 polyacrylamide gel, dried, and analyzed by autoradiography. An example representative of three experiments is shown. The lane at the far left of B represents a sample immunoprecipitated with an isotypematched control antibody.820 A BFold-Change ip-10 Primary RNA Relative to gapdhSHULTZ ET AL.IFN- (h) ip-gapdhIFN- (h)CIFN- (h) 0 0.75 1 2FIG. 2. Ip-10 is transcribed with delayed kinetics due to synthesis of an interferon (IFN)-stimulated regulatory element (ISRE)-binding factor. (A) Wild-type MEFs were treated with 1,000 IU/mL of murine IFN- for various times and ip-10 and gapdh mRNAs were measured by the Northern method. (B) The rate of ip-10 transcription in wild-type MEFs in response to IFN- measured by qPCR, using.T difficult to obtainIRF1 binds to the ip-10 ISREIRF1 is an ISRE-binding protein whose synthesis is induced in response to IFN- (Taniguchi and others 2001). We tested its binding to the ISRE in the ip-10 promoter by supershift analysis (Fig. 3A). Anti-IRF1 supershifted the j.addbeh.2012.10.012 ISRE-binding complex formed in response to a 4-h treatment with IFN- (lane 4). Anti-p65 (an isotype control), on the other hand, did not (lane 5), as expected since p65 binds to B elements, which were not present in the probe. Furthermore, a version of the ISRE probe carrying mutations in the core ISRE-binding domain (GGAAAGTGAAACC GGACAGTGACACC) did not bind at all (lane 3). In the same gel, we also ran samples from cells treated with IFN- for 4 h and then preincubated with a 50-fold excess of unlabeled wild-type ISRE probe, mutant ISRE probe, or GAS probe (lanes 6, 7, and 8, respectively). Only the unlabeled wild-type ISRE probe competed successfully, showing that the complex is specific for that sequence. Finally, two different antibodies that shift Stat1 bound to a GAS probe (data not shown) did not shift the ISRE-bound complex (lanes 9 and 10). These results led us to investigate whether IRF1-null MEFs induce ip-10 in response to IFN-. As shown in Figure 3B, these cells expressed much less ip-10 in response to IFN- after 4 h compared to match wild-type MEFs. The fact that a little IP-10 protein is still induced in these IRF1-null cells suggests partial compensation, perhaps through different IRFs. To determine if IRF1 expression could restore ip-10 induction to wild-type levels, we created an expression construct encoding a variant of the estrogen receptor (ER) fused to IRF1. The rationale is that constitutively expressed IRF1 translocates to the nucleus, causing cellular senescence. On the other hand, the nuclear translocation of IRF1-ER is tightly regulated and, in the absence of estrogen (or an estrogen mimic), it remains in the cytoplasm (Kroger and others 2003). IRF1-null cells expressed very little ip-10 mRNA in response to IFN-, either alone or in combination with tamoxifen (Fig. 3C). Interestingly, when IRF1-null cells stably expressing the IRF1-ER protein were treated with tamoxifenAIFN- (h) -IP: anti-p65 0.5 2 ip-10 ip-INPUTBIP: anti-p65 IKK??IKK+/+ 2 2 ip-IFN- (h)-INPUTip-FIG. 1. Interferon (IFN)- stimulates p65 binding to the ip-10 promoter in an IKK–dependent fashion. (A) Wildtype MEFs or (B) IKK–null MEFs or a pool of the same cells in which IKK- was stably restored were treated with 1,000 IU/mL of murine IFN-. Cross-linked chromatin fragments were immunoprecipitated with anti-p65. The recovered DNA was amplified by PCR with 32P-labeled -dCTP, using primers specific for the promoter region of ip-10. The products were separated in an 8 polyacrylamide gel, dried, and analyzed by autoradiography. An example representative of three experiments is shown. The lane at the far left of B represents a sample immunoprecipitated with an isotypematched control antibody.820 A BFold-Change ip-10 Primary RNA Relative to gapdhSHULTZ ET AL.IFN- (h) ip-gapdhIFN- (h)CIFN- (h) 0 0.75 1 2FIG. 2. Ip-10 is transcribed with delayed kinetics due to synthesis of an interferon (IFN)-stimulated regulatory element (ISRE)-binding factor. (A) Wild-type MEFs were treated with 1,000 IU/mL of murine IFN- for various times and ip-10 and gapdh mRNAs were measured by the Northern method. (B) The rate of ip-10 transcription in wild-type MEFs in response to IFN- measured by qPCR, using.