Analysis of importance of sequences present in -293 to -102 bp ZFP36 promoter fragment. (A) The potential transcription factor binding sites, identified in analyzed promoter fragment by Alibaba 2.1 and TRANSFAC 6.0 programs, are shown. (B) MCF-7 cells were co-transfected with full-length wild type ZFP36 promoter or with mutation in EBS2 (dEBS2), AP-1 (dAP-1) or EBS3 (dEBS3) and 50 ng of pElk-VP16. 24 hrs after transfection luciferase activity was measured. Results are shown as a percentage of activation of mutated variant of the promoter in comparison to the full length -488 to +905 bp fragment, both stimulated with Elk-VP16. Data are presented as mean value ± SD from three independent experiments (**P < 0,01) (C) -292 to -102 bp fragments of ZFP36 promoter originating from different species were analyzed using the Emboss suite of programs. Sequence homologues were found by direct search across EMBL database and pairwase alignment with Smith-Waterman algorithm, using standard parameters (GOP = 10, GEP = 1). Figure shows results of multiple alignment performed with ClustalW2 program (GOP = 10, GEP = 5) and the color-highlighted fragments point the transcription factors binding sites, identified by TESS program working on TRANSFAC6 database, and potentially important for the activation of ZFP36 promoter by Elk-VP16 D) Serum-starved MCF-7 cells were stimulated with EGF (20 ng/ml) or PMA (100 ng/ml) for 2 hrs with or without U0126 pretreatment. Then the cells were harvested and subjected to the Western blot analysis with anti-EGR-1, anti-c-FOS and anti-GAPDH antibodies. (E) The MCF-7 cells were co-transfected with ZFP36 and pExpELK-1 and either EGR-1 targeted siRNA or control siRNA. 18 hrs after transfection serum-starved cells were stimulated with EGF (20 ng/ml) or PMA (100 ng/ml) for 8 hrs. The relative luciferase activity was measured. The bottom panel shows the results of Western blot analysis with anti-EGR or anti-GAPDH antibodies in the lysates collectted from serum-starved MCF-7 cells transfected with EGR-1 targeted siRNA or control siRNA and stimulated 2 hrs with EGF or PMA.
Florkowska et al. BMC Molecular Biology 2012 13:8 doi:10.1186/1471-2199-13-8