Figure 2

Wt-p53 suppresses expression of 14-3-3γ. A. Expression of 14-3-3γ and p53 after γ-irradiation. Cultures of A549, H358 and H322 cells were either left untreated (A549, H358, H322) or exposed to 10 Gy of gamma radiation (A549-10 Gy, H358-10 Gy, H322-10 Gy). Total cell extracts prepared six hours after γ-irradiation and proteins fractionated on SDS-PAGE gels. Protein blots were probed for the presence of 14-3-3γ and p53 using the appropriate antibodies. The β-actin was used as a loading control. Typical results are shown. B. 14-3-3γ protein expression following Ad-p53 infection. A549 cells were grown and then either left untreated (A549) or infected at a multiplicity of infection (MOI) of 10 with a control adenovirus expressing GFP (A549-AdGFP) or with an adenovirus expressing a wt-p53-GFP (A549-Adp53). Seventy two hours after infection the cells were harvested and the presence of 14-3-3γ and p53 detected in total cell proteins by immunoblotting using the appropriate antibodies. β-actin was used as a loading control. C. 14-3-3γ mRNA expression following Ad-p53 infection. The relative levels of 14-3-3γ mRNA expression in samples of panel B were quantified using real-time RT-PCR. The bars depict % change compared with control samples from three different experiments ± SD. Bars showing a significant reduction in message level are marked with an asterisk (p < 0.01 versus Control or Ad-GFP infected). D. 14-3-3γ protein expression following endogenous p53 knockdown by siRNA. A549 cells were cultured and then transfected either with a control non-targeting siRNA (A549-Mock siRNA) or an anti-p53 siRNA (A549-p53siRNA) as described in Materials and Methods. The cells were harvested after 48 h, extracts prepared, and protein blots probed for 14-3-3γ and p53 using the appropriate antibodies. β-actin was used as a loading control.