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Figure 2 | BMC Molecular Biology

Figure 2

From: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

Figure 2

MAD1 promoter reporter gene constructs are regulated by C/EBP transcription factors. A. Schematic representation of the position of the CCAAT box (half-sites) and GC box motifs within the MAD1 promoter region. The major transcriptional start site is indicated with +1. B. and C. HeLa cells were co-transfected with the -1284 to +248 or the -184 to +248 reporter gene constructs, respectively, and plasmids encoding the three indicated C/EBP proteins. The promoter-less pGL2 construct served as negative control and its activity was set as 1. The luciferase measurements were normalized to β-galactosidase activities expressed from a co-transfected plasmid. D. Reporter gene assays were performed in HeLa cells by co-transfecting the -184 to +248 reporter gene construct and a pSuper-based shRNA targeting C/EBPβ or a control shRNA. Luciferase activity was determined 2 days after transfection. E. The efficiency of the shRNA targeting C/EBPβ was examined in HeLa cells by immunoblotting. C/EBPβ-specific antibodies were used to detect the two major isoforms of endogenous C/EBPβ proteins, LAP and LIP, in whole cell extracts. F. The C/EBPβ-mediated relative activation of the indicated MAD1 promoter constructs were analyzed in reporter gene assays performed in HeLa cells as described above.

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