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. 2014 Nov 24;34(33):4300–4310. doi: 10.1038/onc.2014.359

Figure 2.

Figure 2

The aggregation domain in p53 is not required for p63/p73 binding or the ability to promote invasion. (a) Immunoprecipitation of p53 with DO-1 in HCT116 p53−/− cells transfected with 3 μg TAp63α in combination with 3 μg p53wt, p53R175H, p53R175H Δ251–312, p53R175H Δ251–257 or p53R175H I254R. p53 and p63 expression was determined in the inputs and in precipitated material by western blot using DO-1 and BC464 (p63) antibodies. (b) Immunoprecipitation of p53 with DO-1 in HCT116 p53−/− cells transfected with 3 μg HA-tagged TAp73α in combination with 3 μg p53wt, p53R175H, p53R175H Δ251–312, p53R175H Δ251–257 or p53R175H I254R. p53 and p73 expression was determined in the inputs and in precipitated material by western blot using DO-1 and HA antibodies. (c) Immunoprecipitation of p53 with DO-1 in HCT116 p53−/− cells transfected with 3 μg TAp63α in combination with 2 μg p53wt, p53R175H, p53R273H, p53R248W or p53I254R. p53 and p63 expression was determined in the inputs and in precipitated material by western blot using DO-1 and BC464 (p63) antibodies. (d) Immunoprecipitation of p53 in HCT116 p53−/− cells transfected with 3 μg HA-tagged TAp73α in combination with 3 μg p53wt, p53R175H or p53I254R. p53 and p73 expression was determined in the inputs and in precipitated material by western blot using DO-1 and HA antibodies. (e) Immunoprecipitation of p53 (folded: 1620; unfolded: 240) in HCT116−/− cells transfected with 3μg p53, p53R175H, p53R273H or p53I254R. p53 expression was determined in the inputs and in precipitated material by western blot using a DO-1 antibody. (f) H1299 cells were retrovirally infected with an empty vector or the indicated p53 mutants (in a pWZL blast vector). Blasticidin-resistant cells were selected and their invasion towards hepatocyte growth factor (HGF) over 72h was measured in inverted transwell invasion assays. Bars represent the mean of invasion beyond 45 μm in four sets of triplicates. Error bars as standard error of the mean. *A P-value <0.02 compared with the empty vector-expressing cells. The expression of the constructs is shown by western blot and actin was used as a loading control (right). Panels form part of the same gel.