FIG 6.

p53 is reduced in livers of dnp300 mice and is a target for regulation by CUGBP1. (A) Expression of C/EBPβ, C/EBPα, and p53 proteins after injection with CCl₄ was determined by Western blotting. The β-actin control is shown for C/EBPα and p53 membranes. (B) Protein levels of C/EBPα and p53 were calculated as ratios to β-actin levels. (C) Amounts of Ub-C/EBPα conjugates are increased in livers of dnp300 mice, while Ub-p53 conjugates are not detected. C/EBPα and p53 were immunoprecipitated from livers of three dnp300 mice, and the IPs were examined by Western blotting with Abs to ubiquitin. Incubation of beads with proteins from WT and dnp300 mice served as a control for nonspecific absorption. Bottom images show Western blotting of p53 and C/EBPα IPs with the corresponding antibodies to p53 and C/EBPα. (D) Protein levels of CUGBP1 are increased in livers of dnp300 mice compared to levels in livers of WT mice. Bar graphs show levels of CUGBP1 as ratios to the β-actin level. (E) Nucleotide sequence of the 5′ UTR of p53 mRNA (top), RNA probes (middle), and diagram of CUGBP1 constructs (bottom). The binding site for CUGBP1 is shown in blue. (F) UV cross-linking analyses of the interactions of WT and mutant (Mut) p53 probes with CUGBP1 proteins. (Left) Competition of WT and mutant oligonucleotides for binding to WT CUGBP1. (Middle) Interactions of CUGBP1 mutants (shown on the top) with the WT p53 probe. (Right) Bar graphs showing binding of CUGBP1 proteins as ratios to the amounts of proteins determined by densitometry of Coomassie stains.