Skp2B overexpression promotes the accumulation of denatured p53 species.

<p>A) Cycloheximide chase in MCF-7 and MCF-7Skp2B cells was performed for the indicated time and cells were harvested for immunoblotting using anti-p53 antibody. Tubulin was used as a loading control. B) Quantification of the fraction of p53 remaining in MCF-7 and MCF-7Skp2B cells at the indicated time of cycloheximide treatment. C) Proteins from MCF-7, MCF-7Skp2B and T47D cells were used for immunoprecipitation of endogenous p53 using the mouse monoclonal pAb240 anti-p53 antibody, which immunoprecipitates only denatured but not wild-type forms of p53. The immunoblot was then developed using an anti-rabbit p53 antibody. D) Extracts from MCF-7 and MCF-7Skp2B were digested with increasing concentrations of thermolysin and the resulting cleavage products detected by immunoblot using the DO1 antibody. The 27kDa product is indicated. E) MCF-7 and MCF-7-Skp2B cells were treated with or without camptothecin (10 µM) for 4 hours and the levels of GADD45 mRNA levels determined qRT-PCR. The difference between the levels of GADD45 mRNA following DNA damage between MCF-7 and MCF7-Skp2B cells was statistically significant. The three arrows indicate a p value of less than 0.0001. F) MCF-7 and MCF-7-Skp2B cells were treated with or without camptothecin (10 µM) for 4 hours and the levels of PUMA mRNA levels determined qRT-PCR. The difference between the levels of PUMA mRNA following DNA damage between MCF-7 and MCF7-Skp2B cells was statistically significant. The three arrows indicate a p value of less than 0.0001. G) MCF-7 treated with mock siRNA and MCF-7-Skp2B cells were treated with 1 or 2 uM camptothecin for 7 days and the percentage of cells that survived determined. The difference between the percentage of cell surviving following DNA damage between MCF-7 and MCF7-Skp2B cells was statistically significant. The three arrows indicate a p value of less than 0.0001.