Validation of the Notch activity reporter and application for high throughput RNAi. Validation of the m3-luc reporter by RNAi targeting of known Notch pathway components. Notch induced E(spl)m3 signal normalized against A. the control viral promoter or B. uninduced E(spl)m3 promoter transcription. C. Uninduced E(spl)m3 promoter expression normalized by control promoter. For each dsRNA, 32 independent wells were measured in a 384-well plate format. Each box encloses 50% of the data with the median value displayed. The error bars mark the full range excluding the shown outliers. D. Schematic of the automated high throughput screen in 384-well plates. Drosophila Kc167 cells were incubated with a unique dsRNA per well. After a four day incubation, the cells were split into three different transfection mixes in duplicate. Firefly luciferase signals were read 24 h after transfection.