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Figure 6 | BMC Molecular Biology

Figure 6

From: Regulation of p73 by Hck through kinase-dependent and independent mechanisms

Figure 6

Hck inhibits the transcriptional activity of p73α and β isoforms, dependent on its SH3 domain. (A-D) Hela cells transfected with pCMV-βgal (50 ng) and different promoter constructs (100 ng) along with p73α or p73β and Hck or KD-Hck were subjected to either luciferase or CAT activity measurements. The amount of DNA was kept constant in all transfections to 400 ng by adding pcDNA3. Hck, KD-Hck, mSH3-Hck and c-Abl plasmids were used at 100 ng for different promoter constructs. The amount of p73α used was 2 ng for Ipaf-CAT promoter and 50 ng for MDM2-Luc promoter. The amount of p73β used was 10 ng for PG13-Luc promoter. Relative reporter activities were calculated after normalizing with β-galactosidase activities. Data presented here are mean ± S.D. of at least three independent experiments, *p < 0.01.(E) Effect of c-Src on p73 induced Ipaf promoter transctivation. HeLa cells transfected with Ipaf promoter construct along with p73α (2 ng) and c-Src or Hck (100 ng) were subjected to CAT activity measurements. Data presented here are mean ± S.D. of at least three independent experiments, *p < 0.05, **p < 0.01. (F, G) Total RNA isolated from Hela cells transfected with HA-p73α (200 ng) or Hck (1300 ng) and mSH3 Hck (1300 ng) were subjected to RT-PCR analysis for Ipaf, MDM2 and PUMA gene expression. p73α and Hck mRNA levels were determined for transfection efficiency control whereas GAPDH mRNA levels were used as an internal control.

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