Figure 1
From: Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation
Def selectively induced the degradation of p53 and Δ113p53 proteins. (A) Western blot of p53 and Δ113p53 using the A7-C10 monoclonal antibody to detect both proteins in defhi429 homozygotes and non-homozygous siblings at 5 dpf and in γ-ray-treated wild-type embryos. ?, uncharacterized p53 isoforms; β-actin, loading control. (B) Coimmunostaining of Fib and p53/Δ113p53 in a defhi429 mutant embryo injected with st-MO (upper panel), Δ113p53-MO (middle panel) or p53-MOATG (bottom panel) morpholinos at 4 dpf. Nuclei were stained with DAPI. st-MO: standard control morpholino. in: intestine. (C) Western blot (top three panels) of p53 protein and northern blot (bottom two panels) of p53 mRNA in embryos injected with different mRNA mixes at 6 hpi as shown. 28S rRNA: RNA loading control. GAPDH, protein loading control. (D) Same as in (C), but analysis of Δ113p53. (E) Same as in (C), but analysis of EGFP. (F) tp53M214K mutant embryos were injected with different mRNA mixes or phenol red dye. The survival rate of embryos in each treatment group at 12 hpi was analyzed. The values plotted represent mean ± SEM (three repeats of n = 100-200 embryos each), with test P-values indicated. (G) Analysis of apoptosis in embryos described in (F) at 10 hpi. Embryos were categorized based on their number of apoptotic cells. Category 1, < 50 apoptotic cells per embryo; category 2, between 50-300 apoptotic cells per embryo; category 3, > than 300 apoptotic cells per embryo. Graphics shows the number of embryos in each category in each case. (H) qPCR analysis of p53 target genes in embryos described in (F). The qPCR values were normalized against elf1a and expressed as fold change in expression. The values plotted represent mean ± SEM. The P-value was obtained by performing the two-tailed unpaired t-test. ***P < 0.001; **P < 0.01.
