Fig. 2.

Characterization of MDM2 binding to engineered FG loop ¹⁰FN3 domains. (A) Schematic diagram of a direct pull-down assay. (B) Western blot analysis of immunoprecipitated HA-tagged FG loop monobodies and FLAG-tagged MDM2 as shown in (A). Respective in-vitro transcription/translation (IVT) expressed HA-tagged FG loop ¹⁰FN3 domains were first immunoprecipitated with anti-HA antibody coated magnetic protein G beads followed by incubation with IVT expressed FLAG-tagged MDM2. (C) Schematic diagram of a competitive binding assay. (D) IVT expressed FLAG-tagged MDM2 was pulled down with anti-FLAG antibody coated magnetic protein G beads followed by co-incubation with respective IVT expressed HA-tagged FG loop ¹⁰FN3 domains and p53.