Genome-wide functional screen was performed as described previously (25). HEK293T cells were cotransfected with pC99-TetOn, pTRE-GFP, monomeric RFP vector, and either control vector (pCtrl) or each cDNA for 18 hours and then treated with doxycycline (100 ng/ml; Sigma-Aldrich) for another 12 hours. The AICD–reverse Tet-controlled transactivator (rtTA) is generated by endogenous γ-secretase; then, it is transported into the nucleus for inducing GFP expression. The putative positive cDNA clones that markedly increased green fluorescence under fluorescence microscope (Olympus) were isolated. The cDNAs encoding membrane proteins were generated by RT-PCR analysis and subcloned into mammalian expression vectors or purchased from OriGene Inc. (25). After the primary screening, the secondary screening was performed using luciferase reporter assay. HEK293T cells were cotransfected with pC99-GVP, pUAS-luciferase, pβ-galactosidase, and experimental control genes with or without 1 μM Compound E. After 24 hours, cell extracts were analyzed by luciferase assay following the manufacturer’s instructions (Promega). The cDNA library was prepared as previously described (37, 57), and the data are provided in data file S1.

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