Figure 6
Figure 6. NF-κB binds directly to the Btk promoter and induces Btk transcription. (A) The chromatin of A20 cells was cross-linked, sheared, and immunoprecipitated with the indicated antibodies. Input and immunoprecipitated DNA was purified and used as template in PCR with primers specific for the Btk promoter (region from −881 to −500 that contain the 2 putative NF-κB sites). Primers for PU.1 (region from −261 to +91, which contains the PU.1 site) were used as positive control; rabbit normal IgG in lane 2 serves as negative control. (B) Nuclear extracts from A20 cells were prepared and DNA-protein–binding activity was analyzed by EMSA. Labeled oligonucleotide probe (lanes 1-4 and 5-7) for NF-κB site 1 (Figure 4C) is described in “Methods.” A labeled probe was incubated without (lane 1, negative control) or with (lanes 2-7) nuclear protein. One hundred–fold molar excess of unlabeled oligonucleotide (cold probe) was used as competitor (lane 3). Anti-p65 antibody, anti-p50 antibody, and rabbit normal IgG are present in lanes 5, 6, and 7, respectively. The probe was replaced by the mutant probe in lane 4. (C) NMRI mice were transfected with 30 μg 1000-Btk (Btk promoter reporter construct) using the hydrodynamic procedure. In addition, the mouse to the left received 7 nmol mixed oligonucleotide decoys (Wt-1 + Wt-2, oligonucleotides corresponding to the NF-κB–binding sites 1 and 2 elements in Btk promoter; details in “Methods”). The mouse to the right received 7 nmol mixed mutant oligonucleotides (Mu-1 + Mu-2). In vivo biophotonic imaging was performed using the IVIS imaging system as described in “Methods.” Data are representative of 3 independent experiments. (D) The 500-Btk, and the 1000-Btk and its mutant promoter reporter constructs (1000-Btk-Mu 1, 1000-Btk-Mu 2, and 1000-Btk-Mu-(1 + 2)) were cotransfected with p65/RelA plasmid into A20 cells. Forty-eight hours later, luciferase activity was measured as described in “Methods”; relative levels of luciferase activity are shown. Data are representative of 3 independent experiments.

NF-κB binds directly to the Btk promoter and induces Btk transcription. (A) The chromatin of A20 cells was cross-linked, sheared, and immunoprecipitated with the indicated antibodies. Input and immunoprecipitated DNA was purified and used as template in PCR with primers specific for the Btk promoter (region from −881 to −500 that contain the 2 putative NF-κB sites). Primers for PU.1 (region from −261 to +91, which contains the PU.1 site) were used as positive control; rabbit normal IgG in lane 2 serves as negative control. (B) Nuclear extracts from A20 cells were prepared and DNA-protein–binding activity was analyzed by EMSA. Labeled oligonucleotide probe (lanes 1-4 and 5-7) for NF-κB site 1 (Figure 4C) is described in “Methods.” A labeled probe was incubated without (lane 1, negative control) or with (lanes 2-7) nuclear protein. One hundred–fold molar excess of unlabeled oligonucleotide (cold probe) was used as competitor (lane 3). Anti-p65 antibody, anti-p50 antibody, and rabbit normal IgG are present in lanes 5, 6, and 7, respectively. The probe was replaced by the mutant probe in lane 4. (C) NMRI mice were transfected with 30 μg 1000-Btk (Btk promoter reporter construct) using the hydrodynamic procedure. In addition, the mouse to the left received 7 nmol mixed oligonucleotide decoys (Wt-1 + Wt-2, oligonucleotides corresponding to the NF-κB–binding sites 1 and 2 elements in Btk promoter; details in “Methods”). The mouse to the right received 7 nmol mixed mutant oligonucleotides (Mu-1 + Mu-2). In vivo biophotonic imaging was performed using the IVIS imaging system as described in “Methods.” Data are representative of 3 independent experiments. (D) The 500-Btk, and the 1000-Btk and its mutant promoter reporter constructs (1000-Btk-Mu 1, 1000-Btk-Mu 2, and 1000-Btk-Mu-(1 + 2)) were cotransfected with p65/RelA plasmid into A20 cells. Forty-eight hours later, luciferase activity was measured as described in “Methods”; relative levels of luciferase activity are shown. Data are representative of 3 independent experiments.

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