siRNA screen for modifiers of Smad4 nuclear translocation

The TGFb signaling pathway is a critical regulator of developmental processes and disease. We developed a system to measure TGFb signaling dynamics by monitoring the localization of Smad4. We performed an unbiased genome-wide RNAi screen to identify genes involved in ligand-dependent Smad4 nuclear translocation C2C12 GFP-Smad4 reporter cells were transfected with pools of 3 siRNAs targeting individual genes; after 48 hours, cells were stimulated with 2ng/ml TGFb1 for 1 or 6 hours, then fixed and analyzed for Smad4 localization. Two independent experiments were performed, screening 17,582 genes. On each plate, two control wells (siRNA targeting TGFbR1 and XpoI) were included