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Figure 4 | Breast Cancer Research

Figure 4

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

Figure 4

C/EBP-α, C/EBP-β and GATA-1 transactivate the Jab1 promoter. (a) -472/+83 Jab1-Luc reporter plasmid was cotransfected with expression plasmids for C/EBP-α, C/EBP-β, C/EBP-δ, and GATA-1-6. The results are presented as fold activation compared with cells transfected with pcDNA3.1 vector control. All values were compared with the vector control by Student's t test. (b) Left, LAP1 (C/EBP-β1), LAP2 (C/EBP-β2), or LIP (C/EBP-β3) plasmids, along with the -474 Jab1-Luc promoter construct, were cotransfected with the Renilla luciferase plasmid pRL-null (transfection control) into MCF7 cells, and luciferase activity was assayed. Right, western blot analyses to demonstrate the protein expression of LAP1, LAP2 and LIP. (c) Endogenous expression of the C/EBPβ isoforms (LAP1, LAP2, and LIP) and GATA-1 were detected at higher levels in breast cancer cell lines compared with normal mammary epithelial cells by western blot analysis. (d) Identification of proteins in complexes by ChIP assays. Antibodies to β-actin, C/EBP-α, C/EBP-β, or GATA-1 were used to immunoprecipitate protein/DNA complexes from the sonicated lysates of MCF7 cells. After reversing the cross-linking, DNA was precipitated and PCR was performed using primers to amplify the -472/-344 promoter DNA. As a negative control, primers for the upstream promoter region -642/-475 were used. Bottom panel represents quantification of the data with ImageJ software. C/EBP, CCAAT/enhancing binding protein; ChIP, chromatin immunoprecipitation; Jab1, c-Jun activation domain-binding protein-1; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; PCR, polymerase chain reaction; pRL, Renilla luciferase reporter vector; RT-PCR, reverse transcription polymerase chain reaction.

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