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Fig. 2. Binding of 14-3-3 subtypes to CDC25B is site specific. (A) Putative 14-3-3 consensus binding sites in CDC25B. (B-D) Mutants of CDC25B were transfected into HEK293 or U2OS cells either alone or together with 14-3-3 subtypes as indicated. Recovered CDC25B proteins are indicated (upper panel of each set of figures). The letters at the top and numbers at the bottom of each blot represent the CDC25B mutants: wild-type (1); S81A (2); S137A (3); S216A (4); S309A (5); S361A (6); 81S (7); 137S (8); 216S (9); 309S (10); 361S (11); 5SA (12). The definitions of the abbreviations for each mutant are described in the text. (B) Mutants of CDC25B were co-transfected into HEK293 cells with 14-3-3 subtypes ß, {epsilon} or {sigma}. Protein expression was determined by immunoblot. Wild-type or mutant CDC25B proteins were immunoprecipitated with anti-FLAG beads, and CDC25B-bound 14-3-3 was determined in the lysate (Lysate) and the immunoprecipitate [IP: CDC25B ({alpha}-FLAG Ab)]. Separate panel `long exposure' shows 14-3-3 subtype {sigma} after an exposure for 1 hour. (C) Mutants of CDC25B were transfected into HEK293 cells. Recovered CDC25B proteins and CDC25B-bound endogenous 14-3-3ß (endo-14-3-3ß) or endogenous 14-3-3{epsilon} (endo-14-3-3{epsilon}) were detected with specific antibodies in the lysate (Lysate) and the immunoprecipitate [IP: CDC25B ({alpha}-FLAG Ab]. (D) Mutants of CDC25B were transfected into U2OS cells. Recovered CDC25B and CDC25B-bound endogenous 14-3-3{sigma} (endo-14-3-3{sigma}) were detected with specific antibodies in the lysate (Lysate) and the immunoprecipitate [IP: CDC25B ({alpha}-FLAG Ab].(E) Binding of endogenous and transfected 14-3-3 subtypes to CDC25B mutants. ++, well bound; +, detectably bound; ±, faintly bound (could be detected only after long exposure); -, no binding.