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Fig. 9. Hint1 disrupts the Pontin-Pontin interaction. (A) Purified recombinant GST-Pontin and Pontin-His6 fusion proteins were mixed to allow association. Protein complexes were isolated with GSH-agarose beads and subsequently incubated with increasing amounts of Hint1-His6 protein. After a second pull-down, proteins bound to the GSH-agarose beads or in the supernatant were analysed by western blotting with anti-GST, anti-Pontin ({alpha}-23) and anti-Hint1 ({alpha}-21) antibodies. (B) A similar experiment was performed with GST-ß-catenin and Pontin-His6. After formation and isolation of GST-ß-catenin–Pontin-His6 complexes, increasing amounts of Hint1-His6 were added. Precipitated protein complexes and supernatants were analysed by western blotting as described above. Lanes 1,2: control showing that Pontin-His6 and Hint1-His6 does not nonspecifically bind to GST alone. Lanes 4-6: addition of increasing amounts of Hint1-His6 protein. (C) Mapping of the Pontin-binding sites in Pontin (amino acids 214-294) and Reptin (amino acids 218-289).





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