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. Author manuscript; available in PMC: 2012 Sep 9.
Published in final edited form as: Mol Cell. 2012 Jan 26;45(5):587–597. doi: 10.1016/j.molcel.2011.12.025

Figure 2. Physical associations between WTX, p53, and E1B 55K.

Figure 2

(A) Co-immunoprecipitation of Flag-WTX, E1B 55K and p53 in WTX inducible HEK 293-derived F-5 cells. Immunoprecipitation of cellular lysates using anti-Flag, E1B 55K antibodies or control IgG, followed by immunoblotting with antibodies against E1B 55K, p53 or Flag. Arrowheads indicate Flag-WTX-NS (NS) and Flag-WTX-S (S).

(B) shRNA knockdown of p53 does not reduce co-immunoprecipitation of Flag-WTX and E1B 55K in F-5 cells. Arrowheads indicate Flag-WTX-NS (NS) and Flag-WTX-S (S).

(C) WTX and p53 are coimmunoprecipated in U2OS cells, which lack adenovirus E1B 55K. Flag-WTX and TP53 were co-transfected, followed by immunoprecipitation using either anti-Flag or anti-p53 antibodies and immunoblotting. Arrowheads indicate Flag-WTX-NS (NS) and Flag-WTX-S (S).

(D) Mapping WTX-binding domain within p53 by immunoprecipitation-Western blotting in H1299 cells. Flag-WTX and various HA-p53 fragments were transfected into H1299 cells, immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting. The DNA binding domain of p53 binds to WTX (Renilla: non-specific (NS) protein control). The WTX domain involved in binding p53 (aa 561-1135) is shown in Supplementary (Figure S2B).

(E) Mapping WTX-binding domain within E1B 55K. Flag-WTX and various HA-E1B 55K fragments were transfected into H1299 cells, immunoprecipitated using anti-Flag antibody and analyzed by immunoblotting. The central p53 binding domain of E1B 55K binds to WTX (V: empty vector).The WTX domain binding to E1B 55K (aa 561-850) is shown in Supplementary (Figure S2C).