Figure 3.

Mutational analysis of the Sox2-interaction domain in Nanog. (A) Co-immunoprecipitation of endogenous Sox2 and Nanog from E14Tg2a nuclear extract. Immunoprecipitation was performed with Sox2 antibody and immunoblot probed with anti-Nanog or anti-Sox2 antibodies. (B) Co-immunoprecipitation of endogenous Nanog and Sox2 from E14Tg2a nuclear extract. Immunoprecipitation was performed with Nanog antibody and immunoblot probed with anti-Sox2 or anti-Nanog antibodies. (C) Left, schematic representation of the (HA)₃Nanog constructs. Right, co-immunoprecipitations of Nanog variants with Sox2. (FLAG)₃Sox2 and (HA)₃Nanog deletion mutants were transfected into E14/T cells. Immunoprecipitations were performed with an HA antibody as indicated and immunoblots probed with anti-FLAG or anti-HA antibodies. I is 1% of input. (D) Left, schematic representation of the (HA)₃Nanog constructs. Right, co-immunoprecipitations of Nanog variants with Sox2. (FLAG)₃Sox2 and (HA)₃Nanog deletion mutants were transfected into E14/T cells. Immunoprecipitations were performed with an HA antibody as indicated and immunoblots probed with anti-FLAG or anti-HA antibodies. I is 1% of input. (E) Left, Sox2, co-expressed in E. coli with either a Maltose Binding Protein-tryptophan repeat (MBP-WR) fusion protein or a Maltose Binding Protein-tryptophan repeat in which all the tryptophans were mutated to alanine (MBP-WR_W10-A). Right, MBP fusion proteins and associated proteins were purified on amylose resin, subjected to SDS–PAGE and immunoblots probed with Sox2 or MBP antibodies.

Source data for this figure is available on the online supplementary information page.

Source data for Figure 3 ^{(666.8KB, pdf)}