Computational protocol: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

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Protocol publication

[…] Cell lysates were prepared as previously described. For immunoprecipitation, cell extracts were prepared from enriched metaphase cells by mitotic shake-off. Mitotic cell lysates were precleared by incubation with protein-A/Sepharose beads, and then antibodies were applied. Immunoprecipitation of Galectin-1 and incubation of protein-A coated beads alone served as controls. Immunocomplexes were recovered by addition of Protein-A/Sepharose beads overnight at 4 °C, followed by three washes in PBS. Finally, the beads were resuspended in Laemmli buffer for Western blot. For the identification of Galectin-3 binding proteins, beads were submitted to proteolytic digestion and the peptides were analyzed by mass spectrometry (ESI-LTQ-Orbitrap spectrometer) coupled with nano LC chromatographic separation system (Proxeon) in the IJM Proteomic facility. For immunoprecipitation in presence of sugar agonists, glucose, lactose or galactose was added to the lysis buffer to a final concentration of 300, 100 and 300 mM, respectively. Samples were loaded in NuPAGE precast polyacrylamide gels (Thermo Scientific). Primary antibodies were detected using HRP-conjugated secondary antibodies and SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific), visualized on a ImageQuant LAS4000 (GE-Healthcare). Signal quantification was performed using ImageJ. [...] Immunofluorescent images were acquired on a Zeiss Axio Imager.Z1 microscope (Zeiss) equipped with an AxioCam digital camera using a 100x NA 1.4 objective. When applied, deconvolution was performed as previously described. Confocal image sections were acquired on Leica TCS SP5 microscope (Leica Microsystems, Deerfield, Illinois, USA) using a 63x NA 1.4 and 100x NA 1.4 lenses from the Imagoseine plateform (IJM). 3D-SIM images were acquired on the Nikon N-SIM system using a 100x APO TIRF NA 1.49 objective in the NIKON Imaging Center at the Curie Institute (Paris, France). The images reconstruction based on the Gustafsson methods were performed on the NIS-Elements Ar software. Videomicrosocopy were performed with a spinning-disk confocal set-up on a Nikon TiE inverted microscope equiped with a CoolSNAP HQ2 camera operating under Metamorph software. Images were then adjusted for brightness and contrast using ImagJ software.For ciliary length measurement, primary cilia were detected using acetylated-α-tubulin immunostaining. 3D reconstruction of confocal Z-stacks acquired was performed and ciliary length was measured using the filament option of Imaris Software.For the enrichment of Galectin-3 at the spindle, ImagJ was used to manually measure the total pixel intensity of Galectin-3 at the spindle pole, which was reported to the total pixel intensity of Galectin-3 in the cytoplasm. The measurement of NuMA volume along the spindle was performed on ImageJ as previously described. Spindle pole was manually isolated using α-tubulin staining. Line scan was performed for each isolated spindle for α-tubulin and the corresponding marker analyzed. Volume analysis was performed using 3D Object Counter plugin on ImageJ. […]

Pipeline specifications

Software tools ImageJ, MetaMorph, Imaris
Application Microscopic phenotype analysis
Diseases Renal Insufficiency, Chronic