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Supplementary Figure S4. Characterization of the PA2G4-MYCN protein-protein interface. A, BE(2)-C cells were transiently transfected with EV, wildtype PA2G4 or 6 different PA2G4 point mutants for 48 hours, then treated with 100 µg/µl Cycloheximide (CHX) for up to 60 minutes, followed by immunoblot analysis for MYCN protein half-life. B, Differential Scanning Fluorimetry (DSF) showed both the seven amino acid (DHKALST, aa248-254) and large peptide (GGDHKALSTGEDTL, aa246-259) MYCN oligopeptides, along with the MYCN oligopeptide shown not to bind via SPR (DHAALAT) changed the melting temperature of the PA2G4 protein, relative to baseline (i.e. 0mM), in a dose-response manner. Shown are the means of 3 independent experiments {plus minus} SEM. C, An example of the raw data for DHKALST, with the shift to the left correlating to an increase in concentration. D, Raw SPR data for PA2G4 triple mutant (R271A, R272A and S47A) and single mutants (S47A and R272A). Also, raw SPR data for MYCN oligopeptide mutants (DHAALST, DHAALAT and DHKALAT). For the mutants and triple mutations, no binding was observed, thus analysis could not be conducted and is not shown. E, Root Mead Squared Deviation (RMSD) of the peptide over the time of the simulation. After the first 100 frames the peptide is relatively stable. Analysis was only conducted after this time. |
