Figure 5. Functional octamer factor binding sites in the Il6 locus. (A) Organization of the Il6 locus and location of the core promoter region. Positions of four consensus octamer sites identified in silico are shown. AK039125 is an adjacent gene. (B) EMSA analysis on nuclear extracts from 24-h CpG-stimulated splenic B cells, performed using short fragments (160–207 bp) containing the consensus octamer site (ATGCAAT) from an Ig heavy chain promoter or octamer sequences identified in the Il6 locus (site 1, ATTTGCAT −3302; site 2, TTTTGCAT −1438; site 3, ATTTGCAT 3793; site 4, ATTTGCAT 10615). Specific complex formation was detected through supershifts using α-Oct2 or α–OBF-1 monoclonal antibodies, as indicated. Oct2–DNA complexes are indicated with asterisks. The results are representative of three independent experiments. (C and D) Immunoprecipitation (ChIP) of chromatin from purified splenic B cells from mice of the indicated genotypes, using preimmune and hyperimmune rabbit serum specific for Oct2. (C) Cd36 is a known Oct2 target gene (König et al., 1995). (D) ChIP on the same chromatin as in C, but examining the octamer-containing Il6 gene sequences identified in A and positive by EMSA (B). Values in all graphs are means ± SEM (n = 3).
Figure 5.

Functional octamer factor binding sites in the Il6 locus. (A) Organization of the Il6 locus and location of the core promoter region. Positions of four consensus octamer sites identified in silico are shown. AK039125 is an adjacent gene. (B) EMSA analysis on nuclear extracts from 24-h CpG-stimulated splenic B cells, performed using short fragments (160–207 bp) containing the consensus octamer site (ATGCAAT) from an Ig heavy chain promoter or octamer sequences identified in the Il6 locus (site 1, ATTTGCAT −3302; site 2, TTTTGCAT −1438; site 3, ATTTGCAT 3793; site 4, ATTTGCAT 10615). Specific complex formation was detected through supershifts using α-Oct2 or α–OBF-1 monoclonal antibodies, as indicated. Oct2–DNA complexes are indicated with asterisks. The results are representative of three independent experiments. (C and D) Immunoprecipitation (ChIP) of chromatin from purified splenic B cells from mice of the indicated genotypes, using preimmune and hyperimmune rabbit serum specific for Oct2. (C) Cd36 is a known Oct2 target gene (König et al., 1995). (D) ChIP on the same chromatin as in C, but examining the octamer-containing Il6 gene sequences identified in A and positive by EMSA (B). Values in all graphs are means ± SEM (n = 3).

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