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Figure 3 | Breast Cancer Research

Figure 3

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

Figure 3

Functional analysis of the Jab1 promoter. (a) The -472/-312 region contains a regulatory sequence that is sufficient to activate transcription of the Jab1 promoter when cloned in front of the TATA and CAAT boxes. MCF7 cells were transfected with either the -105/+83-Luc or -472/-312, and -105/+83 Jab1-Luc constructs and subjected to luciferase reporter assays. Values were compared with the -105 Jab1-Luc reporter by Student's t test, * P < 0.01. (b) Sequence of the probes used in EMSA assays. The probes span the sequence from -440 to -414 or from -412 to -389 of the Jab1 promoter and encompass the C/EBP or GATA sites. (c) Binding of C/EBP and GATA-1 to the Jab1 promoter. EMSAs showing binding of nuclear proteins C/EBP and GATA-1 to the C/EBP and GATA-1 binding sites of the Jab1 promoter. End-labeled oligonucleotide probes corresponding to the -440/-414 (C/EBP) or -413/-389 (GATA-1) regions of the WT Jab1 promoter sequence were incubated with nuclear extract proteins from MCF7 cells and separated on a 4.5% polyacrylamide gel. Lane 1 shows binding between probe and nuclear extract. Specificity was determined by addition of 50 and 100 molar excess of unlabeled specific cold probe, and mutant probe as specific and nonspecific (NS) competitors as indicated. Lane 4 shows complexes observed in the presence of probes containing mutations in C/EBP or GATA-1. Supershift of the complex was observed following incubation with C/EBPβ or GATA-1 antibodies as shown in the right panels. (d) MCF7 cells were transfected with the -472 Jab1-Luc, -472 C/EBP mutant (Mut1 C/EBP), -472 GATA-1 mutant (Mut 2 GATA1), Mut1+2, or -344 Jab1-Luc plasmids. Mutation of the C/EBP(Mut1C/EBP) and GATA-1 (Mut2GATA1) binding sites reduced Jab1 promoter activity by about 60% and 20%, respectively, and by 75% when mutated together. The firefly luciferase activity of each sample was normalized to that of Renilla luciferase (pRL). Data present the mean ± standard deviation of three independent experiments. All values were compared with the -472 Jab1-Luc reporter by Student's t test. C/EBP, CCAAT/enhancing binding protein; EMSA, electrophoretic mobility shift assay; Jab1, c-Jun activation domain-binding protein-1; NS, nonspecific binding; SS, supershift.

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