(a) Western blot analysis of p53 protein levels in untreated
or doxorubicin-treated (0.2 μg/ml Dox)
p53−/−, p53+/−,
p5325,26,53,54/−, and
p5325,26,53,54/+ MEFs. Actin serves as a loading
control. (b) Western blot analysis of anti-FLAG immunoprecipitation
from p53−/− MEFs transiently
overexpressing HA-p53 and FLAG-p53 or FLAG-p5325,26,53,54. HA-MBP and
FLAG-eGFP were used as negative controls. Immunoprecipitated protein and 10% input
were probed with either anti-HA or anti-FLAG. (μg ratio of HA-p53 to FLAG-p53
or FLAG-p5325,26,53,54 plasmid DNA: 1:1 or 1:2.5). (Supplement to Fig. 3b) (c) Heat map examining the
transactivation capacity of p5325,26,53,54 on p53-dependent genes
identified by microarray analysis through comparison of six
HrasV12;p53 wild-type mouse embryo fibroblast (MEF) lines to six
HrasV12;p53−null MEF lines, as previously
described3. Three independent
HrasV12;p5325,26,53,54/25,26,53,54 MEFs lines were
analyzed, and showed that the gene expression profiles were indistinguishable from
HrasV12;p53 null cells. Numbered columns indicate independent MEF
lines. Blue – repressed genes; Red – induced genes. (d)
qRT-PCR analysis of p53 target gene expression in untreated MEFs derived from
p53+/+ and
p5325,26,53,54/+ E13.5 embryos. Graphs indicate
averages from four independent MEF lines, +/−SD, after normalization to
β-actin. **,*** denote p-values of <0.01, and
<0.005, respectively, by Student’s t-test analysis. (e)
qRT-PCR analysis of p53 target gene expression in p53+/+
and p53−/− MEFs stably transduced with
empty vector, FLAG-p53, or FLAG-p5325,26,53,54. Representative gene
expression from one experiment. +/−SD of technical triplicates after
normalization to β-actin.