Fig. 5: Transactivation potential of constitutively tetrameric TA*p63 and GTAp63 isoforms and its correlation with p300 Taz2 domain interaction. | Cell Death & Disease

Fig. 5: Transactivation potential of constitutively tetrameric TA*p63 and GTAp63 isoforms and its correlation with p300 Taz2 domain interaction.

From: TA*p63 and GTAp63 achieve tighter transcriptional regulation in quality control by converting an inhibitory element into an additional transactivation domain

Fig. 5: Transactivation potential of constitutively tetrameric TA*p63 and GTAp63 isoforms and its correlation with p300 Taz2 domain interaction.The alternative text for this image may have been generated using AI.

a TA assay of C-terminal TA*p63 and GTAp63 isoforms in comparison to TAp63 C-terminal isoforms on the p21 promotor. p53 wt was used as positive, the p53 R175H mutant as negative control. Hundred nanogram of each plasmid (pcDNA3, pGL3 and pRL-CMV) were transiently transfected in Saos-2 cells (12-well plate). Cells were harvested 24 h after transfection and assay was performed. b, c TA titration assay of TA*p63γ and GTAp63γ compared to TAp63γ on the p21 promotor. Hundred nanogram of pGL3 and pRL-CMV and increasing amount of p63 DNA (10 ng, 25 ng, 50 ng, 100 ng, 150 ng) were transiently transfected in Saos-2 cells. Empty vector was added to a total amount of 350 ng DNA for transfection (12-well plate). Cells were harvested 24 h after transfection and assay was performed. Protein levels were determined via western blotting. Fold induction is indicated with white bars, relative protein level with blue colored bars. The protein level of the highest DNA amount of TAp63α was set to 1. Dots represent protein level of each biological triplicate. (d) SDS-PAGE followed by western blotting for myc-tagged TAp63γ, TA*p63γ and GTAp63γ protein level in the TA titration assay performed on p21 promotor in Saos-2 cells. GAPDH was used as loading control. p63 isoforms were detected with the p63 antibody ab124762 (Abcam) for sensitivity reasons. e TA assay of FWL and potential new TA motif mutants to alanine in the N-terminus of the TA*/GTAp63 isoforms (TA*p63: Y18* F22*, W31* Y35*; GTAp63:.W9′ F12′ V15′, W29′ Y33′) on the p21 promotor. p53 wt was used as positive control, the p53 R175H mutant as negative control. Hundred nanogram of each plasmid (pcDNA3, pGL3 and pRL-CMV) was transiently transfected in Saos-2 cells (12-well plate). Cells were harvested 24 h after transfection and assay was performed. Bars represent the mean value of the biological triplicate, error bars represent SD, crosses represent mean value of the technical replicates. f Fluorescence anisotropy experiment with the Taz2 domain of p300 and either N-terminal fluorescein-tagged TA*-GTA*- (1*-39*) or GTA-GTA*- (1′-37′) peptide. A peptide concentration of 100 nM was used for each measuring point with increasing concentration of p300 Taz2 domain

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