Flp in trex

RPE-1 Flp-In T-Rex (a gift from Erich Nigg) and TTBK2 KO cells and RPE1 WT were grown in DMEM F12 (Thermo Fisher Scientific, 11320033) supplemented by 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l-glutamine, HEK293T wt, and TTBK2 KO Flp-In T-Rex (Thermo Fisher Scientific, R78007) cells were grown in DMEM Glutamax (Thermo Fisher Scientific, 10569069) supplemented by 10% FBS and 1% penicillin/streptomycin. Transfection of RPE1 was carried out by Lipofectamine 3000 (Invitrogen) according to manufacturer’s instructions (up to 0.5 µg DNA per condition/well, 24-well plate format). HEK293T were transfected by Polyethyleneimine (PEI) as follows: PEI was incubated in DMEM for 10 min and then mixed with plasmid equilibrated in DMEM in ratio 3 µl PEI/1 µg plasmid; the mixture was incubated for 15 min and then added to cells. Growth medium was changed 4 h after transfection. To induce formation of PC, 24 h following the transfection, RPE-1 cells were starved by serum-free medium for 24 h to assess ciliogenesis or ON for TTBK2 removal measurement. Centrinone (150 µM) (Wong et al., 2015 ) treatment was carried out for 72 h. Cytochalasine (2 µM) (Merck, Sigma Aldrich, C8273) and Nocodazole (100 ng/ml) (Merck, Sigma-Aldrich, M1404) treatment was carried out overnight.

Human NTHL1 was cloned into pcDNA5 FRT/ FLAGBirA* vector. Using the Flp-In™ T-REx™ system (Thermofisher), we generated 293 TRE Flp-In cells stably expressing NTHL1-BirA*-FLAG, eGFP-NLS-BirA*-FLAG, and eGFP-BirA*-FLAG under a tetracycline inducible system. Cells were treated with 1 μg/ml of tetracycline for 24 h to induce protein expression. Six hours before cell collection, 50 μM of biotin were added and immediately followed by irradiation treatment. BioID purification and mass spectrometry data acquisition and analysis were performed as described (47 (link)).