Zebrafish survival in response to simultaneous ectopic expression of all three p53 isoforms in zebrafish embryos. (A) Capped mRNAs encoding FLp53 (0.1 ng mRNA/embryo) alone or in combination with ΔNp53 (1 ng mRNA/embryo) or Δ113p53 (1 ng mRNA/embryo) or ΔNp53 (1 ng mRNA/embryo) together with Δ113p53 (1 ng mRNA/embryo) were injected into 1-4 cell embryos and survival was scored over seven days. Standard error was determined using triplicate dishes of 60 embryos each. Capped mRNA of green fluorescent protein (GFP; 2 ng RNA/embryo) was used as a negative control. (B) Molecular interaction of zebrafish p53 isoforms as determined by coimmunoprecipitation experiments using Saos-2 cells. All immunoprecipitations were performed using the HA Tag IP/Co-IP kit (Pierce, Rockford IL). The immunoprecipitates were subjected to SDS-PAGE and immunoblotted using antibodies recognizing the HA- and Myc-tags. The upper panel shows immunoblot detection of the HA-tag whereas the lower panel shows blots probed with antibodies detecting the Myc-tag. Experimental conditions were as follows: (1) Transfection control, (2) HA-Akt and Myc-ΔNp53 (3) HA-FLp53 and Myc-ΔNp53, (4) HA-FLp53 and Myc-Δ113p53, (5) HA-Δ113p53 and Myc-ΔNp53. The asterisk denotes the HA-Δ113p53 band. Expression of transgenes was validated by Western blot analysis of whole cell extracts (see Additional file 2).
Davidson et al. BMC Developmental Biology 2010 10:102 doi:10.1186/1471-213X-10-102