Figure 4

PR-stable and PR-trans bait cell arrays. 4a: Schematic representation of different transfection strategies tested in CAPPIA. On Prey-Reporter-Bait (PRB) slides, bait and prey expression plasmids AR-LBD and AR-NTD respectively and the reporter plasmid complexed with transfection reagent were immobilized together in array format. In contrast, on PR slides, each spot contained only the reporter and the AR-NTD prey construct. For Prey-Reporter-stable-bait (PR-SB) experiments a HEK293 cell line with a stable integration of the pBD-LBD plasmid was generated and grown on top of the PR slides. For Prey-Reporter-transient-bait (PR-TB) experiments, suspensions of HEK293T cells were incubated with pBD-LBD plasmid complexed with transfection reagent 5 minutes before adding them to the PR-slides. Finally PRB slides were incubated with non-treated HEK293T cells as described before. All cells were cultured on the slides for 3 days in the presence of 10 nM R1881. 4b: When the data are normalised for differences in transfection efficiencies the results show that all three strategies result in a comparable specific trans-activation of reporter expression following AR-LBD and AR-NTD interaction. Data shown represents the mean fluorescence of 6 features per sample, collected from triplicate spots on two identical slides.