Figure 4
Figure 4. C/EBP displaces HDACs from NF-κB p50. (A) Ba/F3 cell lysates were subjected to immunoprecipitation (IP) with the indicated antisera, and coprecipitated NF-κB p50 was detected by Western blotting (WB). (B) NIH-3T3 cells were transiently cotransfected in duplicate with 1 μg of κB×2-Luc, 5 ng of CMV–β-Gal, and the indicated combinations of CMV, CMV-p50, CMV-C/EBPα, or CMV-C/EBPαLZ. TSA or vehicle was added 24 hours after transfection to one set, and luciferase and β-galactosidase activities were determined after an additional 24 hours. Corrected fold activation relative to empty CMV was calculated, and an average from 3 experiments is presented. (C) Ba/F3 cells carrying the indicated ER fusions of C/EBPα variants were cultured without (-) or with (+) estradiol (E2) for 5 hours and subjected to ChIP analysis with the use of antisera for HDAC1 (left) or HDAC3 (right). Enrichment of a DNA fragment corresponding to the proximal or the distal regions of the Flip promoter, determined with quantitative real-time PCR relative to input DNA, in 3 independent ChIP experiments is shown. *P < .001 for the comparison between without (-) and with (+) E2. (D) Parental Ba/F3 cells or clones carrying MT-C/EBPα were cultured with zinc for 16 hours and subjected to ChIP analysis with the use of antisera for C/EBPα (α), HDAC1 (H1), HDAC3 (H3), or normal rabbit IgG (Ig). PCR was used to detect the precipitated FLIP promoter. Endogenous expression of HDAC1 or HDAC3 was determined by Western blotting in similarly treated cells. (E) The indicated Ba/F3 cell clones were cultured with zinc for 16 hours, and chromatin DNA was immunoprecipitated with antibodies against acetyl-histone H3 (AcH3) or acetyl-histone H4 (AcH4). The precipitation of DNA fragments corresponding to the proximal or distal regions of the Flip promoter was determined with the use of quantitative real-time PCR relative to input DNA, and the average from 3 experiments is presented. *P < .01 for the comparison between parental Ba/F3 and cells expressing MT-C/EBPα or MT-C/EBPαLZ.

C/EBP displaces HDACs from NF-κB p50. (A) Ba/F3 cell lysates were subjected to immunoprecipitation (IP) with the indicated antisera, and coprecipitated NF-κB p50 was detected by Western blotting (WB). (B) NIH-3T3 cells were transiently cotransfected in duplicate with 1 μg of κB×2-Luc, 5 ng of CMV–β-Gal, and the indicated combinations of CMV, CMV-p50, CMV-C/EBPα, or CMV-C/EBPαLZ. TSA or vehicle was added 24 hours after transfection to one set, and luciferase and β-galactosidase activities were determined after an additional 24 hours. Corrected fold activation relative to empty CMV was calculated, and an average from 3 experiments is presented. (C) Ba/F3 cells carrying the indicated ER fusions of C/EBPα variants were cultured without (-) or with (+) estradiol (E2) for 5 hours and subjected to ChIP analysis with the use of antisera for HDAC1 (left) or HDAC3 (right). Enrichment of a DNA fragment corresponding to the proximal or the distal regions of the Flip promoter, determined with quantitative real-time PCR relative to input DNA, in 3 independent ChIP experiments is shown. *P < .001 for the comparison between without (-) and with (+) E2. (D) Parental Ba/F3 cells or clones carrying MT-C/EBPα were cultured with zinc for 16 hours and subjected to ChIP analysis with the use of antisera for C/EBPα (α), HDAC1 (H1), HDAC3 (H3), or normal rabbit IgG (Ig). PCR was used to detect the precipitated FLIP promoter. Endogenous expression of HDAC1 or HDAC3 was determined by Western blotting in similarly treated cells. (E) The indicated Ba/F3 cell clones were cultured with zinc for 16 hours, and chromatin DNA was immunoprecipitated with antibodies against acetyl-histone H3 (AcH3) or acetyl-histone H4 (AcH4). The precipitation of DNA fragments corresponding to the proximal or distal regions of the Flip promoter was determined with the use of quantitative real-time PCR relative to input DNA, and the average from 3 experiments is presented. *P < .01 for the comparison between parental Ba/F3 and cells expressing MT-C/EBPα or MT-C/EBPαLZ.

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