Fig. 4: Identification of the PRRX1–TOP2A interaction in human MPNST cells.

a Silver staining after immunoprecipitation of 3xFLAG-PRRX1A. HS-PSS/3xFLAG-PRRX1A cells were treated with 1 µg/mL doxycycline (DOX) and then cell lysates were extracted to immunoprecipitate 3xFLAG-PRRX1A and its associated proteins. The cell lysate of HS-PSS was used to detect nonspecific signals. b Confirmation of the PRRX1-TOP2A interaction by immunoprecipitation. After treating HS-PSS/3xFLAG-PRRX1A/3xHA-TOP2A cells with 1 µg/mL DOX, cell lysates were extracted to immunoprecipitate 3xFLAG-PRRX1A or 3xHA-TOP2A. Input and each immunoprecipitated sample were detected with an anti-FLAG or an anti-HA antibody. c Structure binding prediction of PRRX1A and TOP2A dimer by FoldDock. PRRX1A is shown in pink and TOP2A in green. d Enlarged view of predicted binding sites. Interatomic distances were measured by PyMOL and amino acid residues at possible hydrogen bonding distances were plotted in the stick bond model. Predicted hydrogen bonds are indicated by yellow dotted lines. e Schematic drawing of each 3xFLAG-PRRX1A deletion construct or 3xHA-TOP2A (full-length). Each expression vector was transfected into HEK293T cells and each protein was purified from cell lysates. f Cell-free immunoprecipitation using purified 3xFLAG-PRRX1A deletion constructs and 3xHA-TOP2A (full-length). Purified PRRX1A deletion constructs and 3xHA-TOP2A (full-length) were conjugated in lysis buffer and immunoprecipitation with anti-FLAG antibody was performed.