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Figure 5 | Breast Cancer Research

Figure 5

From: Stat3 and CCAAT/enhancer binding protein beta (C/EBP-beta) regulate Jab1/CSN5 expression in mammary carcinoma cells

Figure 5

Stat3 can synergize with C/EBP-α and C/EBP-β to transactivate the Jab1 promoter. (a) The -472/+83 Jab1-Luc reporter plasmid was cotransfected with expression plasmids for C/EBP-α, or C/EBP-β. Plasmid amounts were normalized by the addition of empty expression plasmid. Following transfection, MCF7 cells were incubated for another 36 hours. The results are presented as fold activation compared with cells transfected with pcDNA vector control. (b) A potential STAT-binding element (TTN5AA consensus sequence) overlaps the C/EBP binding site (CAAC). (c) Binding of Stat3 to the Jab1 promoter is enhanced by the addition of C/EBP. MDA-MB-231 cells were transfected with either control of C/EBP-β2 and collected for ChIP assays using an antibodies against Stat3 and primers for the -472/-344 region of the Jab1 promoter. Right panel represents quantification of the band intensities and normalized to the input with ImageJ software. (d) MDA-MB-468 cells were cotransfected with either pcDNA control, a Flag-tagged Stat3 (Flag-Stat3), or Flag-tagged dominant negative (DN)-Stat3EEE/VV, along with the -472 Jab1-Luc construct, and subjected to luciferase reporter assays. (e) Overexpression of Flag-tagged Stat3 increased Jab1 protein levels while a reduction was seen with Flag-DN- Stat3EEE/VV. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun activation domain-binding protein-1.

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