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Figure 1. Sp1 transcription factor regulates basal and ionomycin-induced GlcAT-I promoter activity. A) HeLa cells were transfected with pGL-302 reporter construct, then treated or not with 1 µM ionomycin in the presence or absence of 1 µM WP 631. Four hours later, cell extracts were prepared, the Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Activity values are mean ±SD of three independent duplicate assays. B) HeLa cells were cotransfected with pGL-302 reporter construct and Sp1 expressing vector pSG-Sp1 or empty vector pSG5 (control). At 24 h post-transfection, cell extracts were assayed for luciferase activity. C) HeLa cells were transfected with pGL-302 construct containing mutated Sp1A (mSp1A), Sp1B (mSp1B), Sp1C (mSp1C), or both Sp1A and Sp1B (mSp1A/B), respectively, then treated or not with 1 µM ionomycin. Four hours after treatment, cell extracts were assayed for luciferase activity. D) For EMSA and supershift assays, 33P-labeled probe from –73 to –46 was used. Lane 1, probe alone; lane 2, probe plus 10 µg of nuclear proteins; lane 3, same as lane 2 plus 100-fold molar excess of unlabeled oligonucleotide; lane 4, same as lane 2 plus 100-fold molar excess of unlabeled Sp1A-mutated oligonucleotide; lane 5, same as lane 2 plus 2 µg of anti-Sp1 Ab; lane 6, same as lane 2 plus 2 µg of anti-Sp3 Ab. The position of specific complex band is marked by an arrow and the supershifted band by an arrowhead. E) For pulldown assay of ionomycin effect on Sp1 DNA binding, 50 mg of nuclear extracts from cells treated or not with ionomycin (1 µM) for 4 h were incubated with biotinylated Sp1A-probe (oligonucleotide –73 to –46) or unrelated probe (control). Sp1A probe-protein complex was then precipitated by addition of streptavidin-agarose beads. The level of Sp1 protein bound to the probe was determined by Western blot using an anti-Sp1 Ab. Equal amounts of nuclear proteins in the different samples were confirmed by using anti-actin antibodies.
