Immunostaining of Cell Lines

A549, H1650, H460, Calu-6, PC9 or AALE cells were plated onto poly-D-lysine-coated eight-well glass chamber slides (5,000 cells per well) for immunostaining. The cells were fixed with 10% buffered formalin and indirect immunofluorescence was performed as described56 (link). Primary antibodies used were phospho-TBK1 (Cell signaling #5483S) or CEP170 mouse monoclonal-72-413-1 (Invitrogen #41-3200) at 1:100 dilution or 1:500 dilution of rabbit monoclonal NuMA (EP3976 Abcam #109262), Alpha tubulin (1:2,000, Sigma #T6074), Dynein intermediate chain (Abcam#ab23905) and Kif2b (Abcam #ab98214 or Novus #NBP86002), γ Tubulin (Santa Cruz #sc51715) were used at 1:100 dilution. Anti-rabbit Alexa Fluor-488 or anti- mouse Alexa Fluor-594 (Molecular Probes) was used as secondary antibody. DAPI (Vector Labs) was used to stain the nuclei. Cells were visualized with a DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope with a × 63/1.40NA oil immersion objective. Images and Z-stacks were produced with three cooled photomultiplier detectors and analysed with the LAS AF software version 1.6.0 build 1016 (Leica Microsystems, Germany).