Figure 4
From: Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation
Def-mediated p53 degradation was inhibited by the cysteine protease inhibitor. (A) Sequencing genomic DNA from different human cells identified two SNPs (G/C at codon position ORF199, A/G at codon position ORF332) in the hu-Def coding region. (B) Western blot of p53, Def, Hdm2, p21 and Bax for comparing the effect of hu-Def encoded by four combinations of (G/C)199 and (A/G)332 SNPs on the stability of p53 in MCF-7 cells co-transfected with MYC-tagged p53 plasmid, and the expression of p53 target genes p21 and Bax at 24 hpt. (C) Same as in (B) but analysis of Δ133p53 in co-transfected cells. (D) Western blot of p53 and Def in MCF-7, HepG2 or Ovcar-8/TR cells treated with def specific siRNA pool (hu-def-siRNA pool) at 24 hpt. (E) Western blot of p53 and Def for comparing the effect of various inhibitors on hu-Def-mediated p53 degradation. (F) Same as in (E) but analysis of the effect of selected inhibitors on Hdm2-mediated p53 degradation. In (D-F), fold changes in expression for p53 or hu-Def against their respective controls (set as 1) are shown under their corresponding panels. GAPDH or β-actin was used as the normalization control.
