Figure 3.

p68 and p72 interact with p53 in vitro and in vivo. (A) Expression of GST vector control and GST-tagged p68/p72 used in the GST pull-down experiments as shown by Western blotting of cell lysates with a GST-specific antibody. (B) GST ‘pull-down' of in vitro-translated (³⁵S-labelled) p53, showing both input and p53 species interacting with GST-tagged p68/p72. (C) Co-IP of p53 and p68 from nuclear extracts. p53 in U2OS extract was immunoprecipitated with the mouse monoclonal antibody (DO-1) and p68 and p53 in the IP were detected by Western blotting with rabbit polyclonal antibodies 2907 (p68) and CM1 (p53). (D) Reciprocal co-IP of p53 and p68. In this case, p68 was immunoprecipitated using the rabbit polyclonal antibody 2907 and immunoprecipitated p68 and p53 were detected by Western blotting with monoclonal antibodies PAb204 (p68) and DO-1 (p53). (E) Co-IP of p53 and p68 using a different p53-specific immunoprecipitating antibody. p53 in U2OS extract was immunoprecipitated with polyclonal antibody (CM1) and p68 and p53 were detected by Western blotting with monoclonal antibodies PAb204 (p68) and DO-1 (p53). (F) Co-IP of p53 and p72. Proteins immunoprecipitated by the p53 antibody (DO-1) (shown in (C)) were also Western blotted for p72 using the rabbit polyclonal antibody K14. (G) Reciprocal co-IP of p53 and p72. p72 was immunoprecipitated with the K14 antibody and p72 and p53 were detected by Western blotting with K14 and DO-1. Note that since only one p72 antibody is available, the same antibody had to be used for IP and Western blotting, giving a strong crossreaction with heavy chain (H). NE: nuclear extract; molecular weight markers (in kDa) are indicated. A nuclear extract from the p53-null cell line SAOS-2 and an irrelevant mouse or rabbit IgG (as appropriate; Cont. IP) were used as controls for IP.