Immunoprecipitation and immunoblotting

Transient transfection of HEK293 cells (ATCC, Middlesex, UK; cell line routinely mycoplasma tested) was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. A total of 3 μg of plasmid DNA was used to transfect cells. Immunoprecipitation and immunoblotting were performed as previously described⁷⁰. In brief, HEK293 cells were lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors. Following centrifugation at 16,200g for 10 min, supernatants were subjected to immunoprecipitation in the presence of 40 μl of Immunosorb A (Medicago, Uppsala, Sweden) overnight at 4 °C on a rotating platform. The following day, the beads were washed 3 × in lysis buffer and after centrifugation the bead pellet was resuspended in 20 μl 4 × Laemmli protein loading buffer and boiled for 5 min at 95 °C. Immune complexes were loaded onto 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Watter and Process, Herentals, Belgium). Membranes were blocked in 10% blocking solution (Rotiblock, Roth, Karlsruhe, Germany) overnight at 4 °C. All primary antibody incubations were performed in blocking buffer for 2 h at RT followed by incubation with fluorescent-labelled anti-mouse antibody for 1 h at RT. The following antibodies were used: mouse anti-V5 (1:2,500, Biozol, Eching, Germany), mouse anti-Flag M2 (1:5,000, Sigma, St. Louis, MI, USA), mouse anti-Tubulin alpha Ab-2 (1:6,000, Thermo Scientific), fluorescent-labelled goat-anti mouse (1:15,000, LI-COR Biosciences, Lincoln, NE, USA). Immunocomplexes were visualized by fluorescence using Odyssey (LI-COR Biosciences) according to the manufacturer’s instruction. Image Studio Lite software v4.0 (LI-COR Biosciences) was used for detection and densitometric analysis of western blot data according to manufacturer’s instructions. Western blot images in Supplementary Fig. 3 have been cropped for presentation. Full size images are presented in Supplementary Fig. 7.