Figure 5.
ΔNp73α regulates mdr1 gene expression in a p53-dependent manner. (a) Analysis of MDR1 luciferase reporter in AGS cells co-transfected with indicated plasmids. ΔNp73α and p53 were flag-tagged to facilitate their detection. Western blot analysis shows equal expression of ΔNp73α and p53 detected with Flag-specific antibody (lower panel). (b) Expression of MDR1 mRNA in the AGS cells transfected with indicated expression plasmids. (c) siRNA directed against p53 increases effect of ΔNp73α on MDR1 transcription. AGS cells were co-transfected with ΔNp73α or pcDNA3 and either small interfering RNA (siRNA) directed against p53 or nonspecific dsRNA. Western blot analysis of ΔNp73α and endogenous p53 in AGS cells (lower panel). (d) ΔNp73α inhibits specific binding of p53 to the MDR1 promoter. Chromatin extracted from AGS cells transfected with indicated plasmids was immunoprecipitated with p53 (DO-1) antibody (lanes 3, 4 and 5), unspecific mouse antibody (lane 2) or without antibody (lane 1) and then amplified by PCR using human MDR1 promoter-specific primers. As an additional control, primers that amplify unrelated DNA (human darpp32 gene) were used (lane 5). Bottom panels show PCR amplification of input DNA. (e) In vivo characterization of the ΔNp73α binding to the MDR1 promoter by chromatin immunoprecipitation (ChIP) assay. The same as (d), except ChIP was performed with anti-flag M2 antibody.