Expression of MDM2 and its transcriptional regulation. A. MDM2 immuno blot in cells with (L and D + Id4) and without Id4 (L-Id4 and D). GAPDH was used as loading control. Representative data from three different experiments is shown. The bottom panel is semi-quantitative analysis of fold change in MDM2 expression relative to LNCaP (L) and normalized to GAPDH (mean ± SEM, *: P < 0.001, compared to L). B. Schematic of MDM2 promoter organization. MDM2 is transcribed from two independent promoters P1 and P2 but both the transcripts are translated from a common start site in exon2. P1 promoter is p53 independent whereas P2 promoter is p53 dependent due to a p53 response element in intron 1 (p53RE). Specific primers were used to determine the transcript abundance of P1 (p53 independent) and P2 (p53 dependent) transcripts. C. P1 and P2 transcript abundance with Real time quantitative PCR analysis in cell lines expressed as fold change from three different experiments in triplicate (mean ± SEM). The expression is first normalized to GAPDH and then to P1 transcript in L and D cells set to 1 (comparison between L and L-Id4 and between D and D + Id4, a: P < 0.001 as compared to P1 transcript b: P < 0.001 compared to P2 transcript). D. Chromatin immuno-precipitation assay demonstrating the binding of p53 to its respective response element in the MDEM2 P2 promoter (intron 1). Data is expressed as mean + SEM of three different experiments performed in triplicate (mean + SEM, *: P < 0.001).