Generation of stable Flp-In T-Rex cell lines

The Flp-In T-Rex U2OS or HEK 293 cells were transfected with the N- or C-terminal GFP-tagged FAM83A–H or GFP alone packaged into a pcDNA5-FRT/TO vector, together with the Flp recombinase pOG44 (Invitrogen) in a ratio of 1 μg:9 μg as described previously (2, 46). Briefly, plasmids were diluted in 1 ml of Opti-MEM (Gibco), 20 μl of PEI (1 mg/ml) was added, and the mix was vortexed and left at room temperature (RT) for 15 min and added dropwise to a 10-cm dish of target cells in 10 ml of complete medium. Twenty-four hours after transfection, cells were selected in media containing hygromycin (50 μg/ml) and blasticidin (15 μg/ml). Resistant cells were grown up to confluency, tested for doxycycline-induced expression of GFP-tagged proteins, and used in subsequent experiments.