Feline Lindhout

132 4 BSA, and washed three times with PBS-T after each antibody incubation step. Proteins resolved on the membranes were visualized using Odyssey Infrared Imaging (Li-Cor Biosciences) or enhanced chemiluminescence. Pull-down assays and mass spectrometry analysis For biotin–streptavidin pull-down assays, HEK293T cells were co-transfected with BirA, BioGFP(-fusion) plasmids (used as bait), and an additional plasmid (used as prey). After ~ 24 h, cells were washed once with ice-cold PBS, harvested in ice-cold PBS supplemented with 0.5× complete protease inhibitor cocktail (Roche), and centrifuged (5 min, 300 g, 4°C). Cell pellets were resuspended in ice-cold lysis buffer (100 mM Tris, 150 mM NaCl, 1% Triton, pH 7.5) supplemented with 1× complete protease inhibitor cocktail, incubated for 10 min on ice, and centrifuged (5 min, 20,000 g, 4°C). Supernatant was used for the binding assay and for generating input samples by boiling for 5 min at 100°C in SDS sample buffer. Beads were pretreated before the binding assay. For regular pull-down assays with cell culture extracts, magnetic Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) were prewashed once with normal washing buffer (20 mM Tris HCl, 150 mM KCl, 0.5% Triton, pH 7.5), incubated for 30 min at room temperature with blocking buffer (20 mM Tris, 150 mM KCl, 0.2 μg/μl CEA, pH 7.5), and washed twice with normal washing buffer. Binding of HEK293T cell lysates and beads was performed for 1 h at 4°C. Beads were subsequently washed five times using normal washing buffer and boiled for 5 min at 100°C in lysis buffer with SDS sample buffer to elute proteins and generate pull-down samples. Alternatively, for pull-down assays with whole brain extracts for mass spectrometry analysis, beads were prewashed twice with low salt buffer (100 mM KCl, 0.1% Triton X-100, 20 mM Tris, pH 7.6), twice with high salt buffer (500 mM KCl, 0.1% Triton X-100, 20 mM Tris, pH 7.6), and twice again in low salt buffer. Binding of HEK293T cell lysates and beads was performed for 1 h at 4°C in presence of whole rat brain extract (prepared as described above), and beads were subsequently washed five times using normal washing buffer. Mass spectrometry analysis of samples was conducted as described before (Cunha-Ferreira et al, 2018). All the mass spectrometry proteomics data have been deposited to the Pride database (http://www.ebi . ac.uk/pride) with the dataset identifier PXD014534. Immunofluorescence staining Cells were fixed for 10 min in 4% formaldehyde and 4% sucrose (neurons) or in 4% formaldehyde (COS7 cells) at room temperature and washed three times with PBS. Fixed neurons were sequentially incubated with primary and secondary antibodies diluted in GDB (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4). Fixed COS7 cells were first permeabilized for 10 min in PBS with 0.1% Triton-X, blocked for 30 min in PBS with 2% BSA, and sequentially incubated with primary and secondary antibodies diluted in PBS with 2% BSA. Cells were washed three times with PBS after each antibody incubation step.