Mouse anti gfp primary antibody

Detection of SDS-insoluble ataxin-3 species was performed as previously described (Pereira Sena et al., 2021 (link)). In brief, 3–6 μg of protein from cell homogenates were diluted in 1× DPBS containing 2% (w/v) SDS and 50 mM DTT. Then, samples were heat-denatured at 95°C for 5 min and filtered through a nitrocellulose membrane (0.45 μm, Cytiva) using a Minifold^® II Slot Blot System (Schleicher & Schüll, Dassel, Germany). Membranes were incubated with the mouse anti-GFP primary antibody (1:1000, sc-9996, Santa Cruz Biotechnology, Inc., Dallas, TX, US) at 4°C overnight and goat anti-mouse 800CW secondary antibody (1:5000, LI-COR Biosciences) at room temperature for 1 h. Fluorescence signals were detected using the LI-COR ODYSSEY FC and quantified with Image Studio 4.0 software (both LI-COR Biosciences).

For pull-down assays on HEK293T cells, cells were grown in 10 cm dishes in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin, and transfected with EGFP, IgSF8-EGFP, or Tenascin-R-EGFP expression constructs using Fugene6 (Promega). Twenty-four hours after transfection, the media was changed to OptiMEM (Invitrogen) for 2 h. Cells were then lysed in 1 ml ice-cold RIPA buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and protease inhibitors (Roche)) for 1 h at 4 °C on a rocking platform. Lysates were spun at 17,000 × g for 30 min at 4 °C. Three µg of human Fc, IgSF8-Fc, or Tenascin-R-Fc was added to 1 ml of supernatant and rotated overnight at 4 °C. Protein-A agarose beads (50 µl slurry) were added and rotated for 1 h at 4 °C. Beads were washed three times in cold RIPA buffer and once in PBS, boiled in 50 µl 2× sample buffer, and analyzed by western blotting using a mouse anti-GFP primary antibody (1:1000; Santa Cruz) and HRP-conjugated goat anti-mouse IgG secondary antibody (1:10,000; Thermo-Fisher). Uncropped and unprocessed scans are provided in Source data file.