Anti lrrc37a antibody

Frozen substantia nigra tissue was selected from the same Control (N = 3), PSP (N = 3) and PD (N = 3) cases used for OPAL multiplex immunofluorescence. Protein lysates were generated using cell lysis buffer (NEB) and brief sonication on ice, followed by centrifugation to pellet insoluble material. Co-immunoprecipitation was carried out using the Dynabeads Protein G immunoprecipitation kit (ThermoFisher Scientific), with an anti-α-synuclein antibody (Abcam) as bait. Proteins bound to beads were eluted and assayed by western blot (as described above) and probed with an anti-LRRC37A antibody (ThermoFisher Scientific). Whole protein lysate and IgG only controls were run on the same blots.

Membrane and cytosolic proteins were isolated from HEK293T cells, iPSC-derived neurons and iPSC-derived astrocytes using the MEM-PER Plus Membrane Protein Extraction Kit (ThermoFisher Scientific), and protein concentrations were determined by bicinchoninic acid (BCA) assay (ThermoFisher Scientific). For western blotting, protein fractions were subject to SDS-PAGE electrophoresis through BOLT Bis–Tris gels (ThermoFisher Scientific) and were blotted onto nitrocellulose membranes. Membrane fractions were confirmed by labelling with an anti-pan-Cadherin antibody (Cell Signaling Technology), and cytosolic fractions were confirmed by labelling with anti-HSP90 (Cell Signaling Technology). Membranes were stripped using Restore plus western blot stripping buffer and re-probed with an anti-LRRC37A antibody (ThermoFisher Scientific).