Protein purification and electrophoretic mobility shift assay (EMSA)

For recombinant protein purification, constructs that correspond to mouse RBPJ (aa 53–474), mouse N1ICD (aa 1744–2113), human MAML1 (aa 1–280), fly Su(H) (aa 98–523), fly NICD (aa 1763–2412) and fly Mastermind (aa 87–307) were expressed and purified from bacteria using a combination of affinity (Ni-NTA or Glutathione), ion exchange, and size exclusion chromatography as previously described (Friedmann et al., 2008). Purified proteins were confirmed by SDS-PAGE with Coomassie blue staining and concentrations were measured by absorbance at UV280 with calculated extinction coefficients. EMSAs were performed as previously described using native polyacrylamide gel electrophoresis (Uhl et al., 2016; Uhl et al., 2010). Proteins concentrations for each gel are listed in figure legends. Acrylamide gels were imaged using the LICOR Odyssey CLx scanner.