rom ATCC/LGC (Germany). U2OS T-REx have been obtained from LifeTechnologies/Thermo Scientifc (Switzerland). Hela cells stably expressing GFP-tagged hRUVBL1 (Hela TDS), mRuvBL1, mRuvBL2 and ANLN (Hela Kyoto) had been kindly offered by Ina Poser, MPI-Dresden. Cells were grown in D-MEM (Glucose/NaPyruvate), 10% FCS and Penicillin/Streptomycin as well as the respective selective antibiotics under 5% CO2 at 37. GFP-RUVBL1, GFP-RuvBL2 and GFP-ANLN expressing cells had been grown in 400 g/ml G418 (Geneticin, Invitrogen, 1013119). For live microscopy, the cells were grown on LabTek chambered coverslips (Nunc). For mitotic arrest, cells have been treated with 0.three g/ml nocodazole for 16 h as well as the loosely-attached cells had been gently shaken off. The cell cycle profile was verified by flow cytometry. For double thymidine block and mitotic enrichment with nocodazole, cells have been seeded 24 h prior therapy. Thymidine (1 mM) was added for 16 h, cells were released for 8 h and treated a second time with thymidine for 16 h. 5 hours upon 
release from the second thymidine block, nocodazole (100 ng/ml) was added for 5 h.
Cells were seeded on cover slips and fixed with methanol for 15 min at -20 or with three.7% formaldehyde/PBS for 15 min at four followed by permeabilization in 0.2% Triton X-100/PBS for 5 min at 4, and processed as previously described [50]. Pictures were taken on an Olympus IX81 fluorescence microscope, applying a 60xOil/1.4/Ph objective (PlanApo, Olympus), and acquired with a CCD camera (Orca AG, Hamamatsu) employing cellR application (Olympus). The antibodies were anti-RUVBL1 (goat polyclonal sc-15259, Santa Cruz, 1:200), anti-RUVBL2 (rabbit polyclonal, generous gift of Irina Tsaneva, UCL London, 1:75), anti-T239 (rabbit polyclonal, custom-made by Eurogentec, 1:200), anti-PLK1 (mouse monoclonal P-5998, Sigma, 1:200), anti-FLAG (mouse monoclonal F-3165, Sigma, 1:1000) and anti-Tubulin (mouse monoclonal T-4026, Sigma, 1:200), anti-Cyclin A (Benzocaine sc-596, Santa Cruz, 23200243 1:one hundred) and anti-GFP (rabbit polyclonal Ab 290, Abcam, 1:1000). DNA was counterstained with DAPI.
Confocal reside imaging was performed on a customized Zeiss LSM 510 Axiovert microscope working with a 63x, 1.four N.A. Oil Plan-Apochromat (Zeiss). The microscope was equipped with piezo concentrate drives (Piezosystem Jena), custom-designed filters (Chroma), and EMBL incubation chambers (European Molecular Biology Laboratory), giving a humidified atmosphere at 37 with 5% CO2 all through the experiment. Sample illumination was typically kept to a minimum and had no adverse impact on cell division and proliferation. Automated multi-location time-lapse movies and reflection-based autofocus on the LSM510 had been controlled by in house-developed computer software depending on macros as previously described [51]. Photos had been analysed with Zeiss LSM510 software.
Cells had been split 24 h following transfection and treated with nocodazole 48 h just after transfection for 16 h, even though a single fraction was left untreated. Cells were lysed in NP-40 buffer (50 mM Tris-HCl pH eight.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, full protease inhibitor (Roche), 1 mM NaF, 1 mM PMSF, 1 mM Na-vanadate) and 1 mg of protein extract was incubated with three g anti-FLAG antibody or 30 l supernatant of the 12CA5 hybridoma cell line generating anti-HA antibody. Immunoprecipitation was performed as previously described [52]. For GFP-tagged protein immunoprecipitation, 1 mg protein extract was incubated with 20 l GFP-trap (Chromotek) and processed following the manufacturer’s protocol.
HeLa cells have been transfected (Olig
