Figure 3
Figure 3. Identification and functionality of a new STAT-binding site in the LMP-1 promoter. (A) Schematic drawing shows the structure of the LMP-1 promoters and the conservation of its 3 STAT-binding sites among different EBV isolates. (B) DNA-binding activity of STAT6 analyzed in nuclear extracts prepared from IL-4–treated or nontreated KMH2-EBV cells by electrophoretic mobility shift assay (EMSA) experiment with the use of an oligonucleotide probe with a STAT6-binding site from the human GL-ϵ promoter. In the supershift experiments the nuclear extracts were preincubated with anti-STAT6 or anti-STAT5 antibodies, whereas in the cold competition experiments they were preincubated with 100-fold molar excess of unlabeled GL-ϵ or LRS-STAT6 probes. (C) DNA-binding activity of STAT6 analyzed in nuclear extracts prepared from IL-4–treated or nontreated KMH2-EBV cells by EMSA with the use of the GL-ϵ– or the LRS-STAT6–labeled probes. In the cold competition experiments, the nuclear extracts were preincubated with 100-fold molar excess of unlabeled GL-ϵ, LRS-STAT6, LRS-STAT6mut1, or LRS-TR probes. (D) DNA-binding activity of STAT6 analyzed in nuclear extracts prepared from IL-4–treated or nontreated KMH2-EBV cells by EMSA with the use of the GL-ϵ–labeled probe. In the cold competition experiments the nuclear extracts were preincubated with 100-, 30-, or 10-fold excess of unlabeled GL-ϵ or LRS-STAT6 probes. In additional competition experiments the extracts were incubated with 100-fold excess of unlabeled LRS-STAT6NPC, LRS-STAT6mut1, LRS-STAT6mut2, LRS-EDL1 or LRS-TR probes. (E) Relative luciferase activity (RLA) of the L428 and L1236 cell lysates after transient transfection with LRS-634 and LRS-STAT6mut1 reporter vectors compared with the pGL3 control. The error bars indicated ± SD of 3 independent experiments. (F) Chromatin immunoprecipitations (ChIPs) were performed with the untreated or IL-4–treated (1 hour, 50 ng/mL) KMH2-EBV cells with the use of polyclonal rabbit control Ig or STAT6-specific antibodies. The results are expressed as the fold enrichment of precipitated DNA after ChIP with anti-STAT6 antibody (gray bars) relative to ChIP with control IgG (white bars) as measured by quantitative polymerase chain reaction with primers located around the STAT6-binding site in the LMP-1 promoter or at 2 negative control regions in the EBV genome (BCLF1 and BALF2). The error bars indicate SD of 3 quantitative polymerase chain reaction measurements of 1 ChIP experiment. The results are representative of 2 independent ChIPs.

Identification and functionality of a new STAT-binding site in the LMP-1 promoter. (A) Schematic drawing shows the structure of the LMP-1 promoters and the conservation of its 3 STAT-binding sites among different EBV isolates. (B) DNA-binding activity of STAT6 analyzed in nuclear extracts prepared from IL-4–treated or nontreated KMH2-EBV cells by electrophoretic mobility shift assay (EMSA) experiment with the use of an oligonucleotide probe with a STAT6-binding site from the human GL-ϵ promoter. In the supershift experiments the nuclear extracts were preincubated with anti-STAT6 or anti-STAT5 antibodies, whereas in the cold competition experiments they were preincubated with 100-fold molar excess of unlabeled GL-ϵ or LRS-STAT6 probes. (C) DNA-binding activity of STAT6 analyzed in nuclear extracts prepared from IL-4–treated or nontreated KMH2-EBV cells by EMSA with the use of the GL-ϵ– or the LRS-STAT6–labeled probes. In the cold competition experiments, the nuclear extracts were preincubated with 100-fold molar excess of unlabeled GL-ϵ, LRS-STAT6, LRS-STAT6mut1, or LRS-TR probes. (D) DNA-binding activity of STAT6 analyzed in nuclear extracts prepared from IL-4–treated or nontreated KMH2-EBV cells by EMSA with the use of the GL-ϵ–labeled probe. In the cold competition experiments the nuclear extracts were preincubated with 100-, 30-, or 10-fold excess of unlabeled GL-ϵ or LRS-STAT6 probes. In additional competition experiments the extracts were incubated with 100-fold excess of unlabeled LRS-STAT6NPC, LRS-STAT6mut1, LRS-STAT6mut2, LRS-EDL1 or LRS-TR probes. (E) Relative luciferase activity (RLA) of the L428 and L1236 cell lysates after transient transfection with LRS-634 and LRS-STAT6mut1 reporter vectors compared with the pGL3 control. The error bars indicated ± SD of 3 independent experiments. (F) Chromatin immunoprecipitations (ChIPs) were performed with the untreated or IL-4–treated (1 hour, 50 ng/mL) KMH2-EBV cells with the use of polyclonal rabbit control Ig or STAT6-specific antibodies. The results are expressed as the fold enrichment of precipitated DNA after ChIP with anti-STAT6 antibody (gray bars) relative to ChIP with control IgG (white bars) as measured by quantitative polymerase chain reaction with primers located around the STAT6-binding site in the LMP-1 promoter or at 2 negative control regions in the EBV genome (BCLF1 and BALF2). The error bars indicate SD of 3 quantitative polymerase chain reaction measurements of 1 ChIP experiment. The results are representative of 2 independent ChIPs.

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