FIGURE 1. TPA induces MnSOD gene transcription.

A, MnSOD gene transcription was measured as luciferase activities from MnSOD promoter- and enhancer-driven pGL3 reporter vector. B, Northern analysis was performed using a human MnSOD cDNA probe. Arrows point to the 1- and 4-kilobase human MnSOD transcripts. The same membrane was stripped and re-probed with β-actin cDNA probe as an internal control (bottom arrow).DMSO,Me₂SO. C, Western analysis of MnSOD in whole cell extracts after TPA treatment. The membranes were stripped and re-probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as an internal control. D, Western analysis of p50, p65, NPM, C/EBP-β, and Sp1 was performed for nuclear extracts. The same membrane was re-probed with β-actin antibody for the loading control. E, EMSA was carried out as described under “Experimental Procedures.” For supershift experiments EMSA reaction mixtures were incubated with 1 µg of antibodies specific to p50, p65, and Sp1. The arrows point to protein-DNA complexes and supershift complexes. Panel a, complexes of NF-κB and DNA were supershifted by antibodies specific to p50 and p65;panel b,Sp1-DNA complexes were super shifted by the antibody specific to Sp1. Each data point represents the mean of three independent experiments %S.D. Significant difference compared with the control: **, p<0.01.