Figure 6.
Figure 6. Analysis of ICSBP's binding to DNA and protein-protein interaction. (A) M1 cells cultivated in the presence of IFNβ were transiently transfected with either the pGL3-Basic vector or the pGL3-Basic -444/-117 promoter construct and cotransfected with expression plasmids for wild-type ICSBP, the DNA binding domain (DBD) mutant ICSBP K79E, the interaction domain (IAD) mutant ICSBP R289E, or the corresponding empty vector and analyzed for luciferase activity 24 hours following transfection. Shown are relative values normalized for Renilla luciferase activity. Error bars depict standard error. (B) M1 cells cultivated in the presence of IFNβ were transiently transfected with the pGL3-Basic -444/-117 promoter construct or a construct harboring a mutation in a potential ICSBP binding site at position -347 bp, pGL3 Basic (-444/-117) Δ ICSBP. An expression plasmid for ICSBP or the corresponding empty vector was cotransfected .The samples were analyzed for luciferase activity 24 hours following transfection. Shown are relative values normalized for Renilla luciferase activity. Error bars depict standard error. (C) EMSA analysis was performed using nuclear extracts (NEs) from M1 cells and radiolabeled oligonucleotides spanning the Ink4b promoter region from -364 bp to -312 bp (Probe C). The -320 bp PU.1 binding site, as well as the potential ICSBP binding site at -360 bp, are present in this region. For lanes 2 to 7, nuclear extract from cells that were cultivated in the presence of IFNβ for 16 hours was incubated with the following oligonucleotide probes: wild-type (WT; lanes 1-4), a probe carrying a mutation in the PU.1 site (PU.1mu probe; lane 5), a probe carrying a mutation in the ICSBP binding site (ICSBPmu probe; lane 6), and a probe carrying mutations in both the PU.1 and ICSBP binding site (PU.1mu/ICSBPmu probe; lane 7). Two specific complexes were resolved. Cold competitor oligonucleotide (CC) at 50-fold excess compared with the labeled oligonucleotide was included for lanes 3 and 4, using either unlabeled wild-type (WT) probe (lane 3) or a probe carrying a mutation (MU) in the PU.1 binding site at -320 bp as well as in the potential ICSBP binding site at -360 bp (lane 4). Arrowheads indicate protein/DNA complexes proposed to contain PU.1 and ISCBP.

Analysis of ICSBP's binding to DNA and protein-protein interaction. (A) M1 cells cultivated in the presence of IFNβ were transiently transfected with either the pGL3-Basic vector or the pGL3-Basic -444/-117 promoter construct and cotransfected with expression plasmids for wild-type ICSBP, the DNA binding domain (DBD) mutant ICSBP K79E, the interaction domain (IAD) mutant ICSBP R289E, or the corresponding empty vector and analyzed for luciferase activity 24 hours following transfection. Shown are relative values normalized for Renilla luciferase activity. Error bars depict standard error. (B) M1 cells cultivated in the presence of IFNβ were transiently transfected with the pGL3-Basic -444/-117 promoter construct or a construct harboring a mutation in a potential ICSBP binding site at position -347 bp, pGL3 Basic (-444/-117) Δ ICSBP. An expression plasmid for ICSBP or the corresponding empty vector was cotransfected .The samples were analyzed for luciferase activity 24 hours following transfection. Shown are relative values normalized for Renilla luciferase activity. Error bars depict standard error. (C) EMSA analysis was performed using nuclear extracts (NEs) from M1 cells and radiolabeled oligonucleotides spanning the Ink4b promoter region from -364 bp to -312 bp (Probe C). The -320 bp PU.1 binding site, as well as the potential ICSBP binding site at -360 bp, are present in this region. For lanes 2 to 7, nuclear extract from cells that were cultivated in the presence of IFNβ for 16 hours was incubated with the following oligonucleotide probes: wild-type (WT; lanes 1-4), a probe carrying a mutation in the PU.1 site (PU.1mu probe; lane 5), a probe carrying a mutation in the ICSBP binding site (ICSBPmu probe; lane 6), and a probe carrying mutations in both the PU.1 and ICSBP binding site (PU.1mu/ICSBPmu probe; lane 7). Two specific complexes were resolved. Cold competitor oligonucleotide (CC) at 50-fold excess compared with the labeled oligonucleotide was included for lanes 3 and 4, using either unlabeled wild-type (WT) probe (lane 3) or a probe carrying a mutation (MU) in the PU.1 binding site at -320 bp as well as in the potential ICSBP binding site at -360 bp (lane 4). Arrowheads indicate protein/DNA complexes proposed to contain PU.1 and ISCBP.

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