 Solution

Include both specific antibodies in the same electrophoretic mobility shift assay and compare with each single-antibody reaction and isotype controls. An additional double supershift of one defined complex would support simultaneous occupancy, especially when reproduced with purified TCF transcription factor, HNF1β and one probe containing the paired sites. Mutating each site separately tests the relevant binding requirement. Failure to obtain a double supershift is not decisive because antibody binding may be sterically incompatible or disruptive. For the native promoter, sequential chromatin immunoprecipitation with the two factors, with reciprocal order and background controls, can test co-association with the same promoter-containing chromatin fragment; it does not alone prove direct contact or base-specific binding.