Figure 2
TAp73β binds to the IER3 promoter and regulates IER3 expression.
(A) The sequences of the IER3 probe encompassing the p53-binding element used to generate radiolabeled double-stranded oligonucleotides are shown. EMSA was performed using nuclear extracts (5 µg) isolated from HeLa cells overexpressing HA-TAp73β or -p53. No nuclear extract was incubated in lane 1. For cold probe, a 200 times excess of unlabeled oligonucleotides were used. Overexpression of HA-TAp73β or -p53 proteins and efficient nuclear subcellular fractionation were determined by immunoblotting using the indicated antibodies. (B) Both HeLa and 293T cells were transfected with plasmids encoding HA-TAp73β or -p53. The ChIP assay was performed using IER3-specific primers that targeted the p53-binding element and quantitative real-time RT-PCR results are shown as enrichment in fold. As a negative control, control IgG was used for immunoprecipitation. Asterisks indicate significant values compared with the control and the results are from three independent experiments run in duplicate (p < 0.05). HeLa cells were transfected with plasmids encoding HA-TAp73β or -p53 (C) and sip73 (D) for 24 h and cell lysates were prepared and immunoblotted with indicated antibodies. Quantitative analyses of the IER3 protein levels induced by TAp73β were shown as the means ± SEM of three independent experiments (bottom panel). GAPDH was used as an equal loading control. (A, C and D) These full-blots membrane was cut into pieces according to estimated molecular weight of proteins of interest and probed with indicated antibodies. All cropped bolts have been run under the same experimental condition.
