Glun1

Mice hippocampi and cerebellums were collected and lysed with ice cold lysis buffer supplemented with 1% protease inhibitor cocktail (Pierce) and phosphatase inhibitor (100 uM NaF and 0.5 mM active Sodium orthovanadate). Protein concentrations were determined by Pierce BCA protein assay kit (Thermo Scientific), and the same amount of total protein was loaded to each lane. The quantification of target protein in each lane was normalized to the total amount of protein, which is determined by Ponceau S staining. Antibodies used included GluA1, p-GluA1, GluN1, PSD95 (all from Cell Signaling), and synaptophysin (Millipore).

BMDMs were transferred to SFM for 30 min and then treated with 10 nM EI-tPA, 10 nM α₂M or vehicle for 1 h. Cells were washed three times with PBS, gently lifted from the plate, and then incubated with 2 mM EZ-Link Sulfo-NHS-LC-Biotin reagent (Thermo Fisher Scientific) for 30 min at 4 °C. This plasma membrane-impermeable reagent labels only the ectodomains of plasma membrane proteins. To quench biotinylation reactions, cells were extensively washed with 20 mM sodium phosphate, 150 mM NaCl, 100 mM glycine, pH 7.4. Cells were then extracted in 1% Triton X-100 containing protease inhibitors for 30 min at 4 °C. Extracts were centrifuged at 12,000× g for 20 min at 4 °C. Supernatants were collected and referred to as the Triton X-100-soluble fraction. The Triton X-100-insoluble pellets were re-extracted in RIPA buffer containing protease inhibitors for 30 min at 4 °C, then centrifuged at 12,000× g for 20 min at 4 °C. Biotinylated cell surface proteins from both fractions were affinity precipitated with Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific). Precipitates were subjected to SDS-PAGE and immunoblot analysis to detect LRP1 β-chain (Abcam), LRP1 α-chain (Sigma-Aldrich), GluN1 (Cell Signaling Technology), and PrP^C (POM19, a monoclonal antibody which is previously described^{76 (link)}). Uncropped blots are presented in Supplementary Figures online.

Supernatants containing the total proteins (added with loading buffer, boiled at 100°C for 5 min) or biotinylated proteins were loaded into 7.5% Bis-Tris gels (Bio-Rad, Hercules, CA, USA). After running in 1× NuPAGE MOPS SDS buffer (Fisher Scientific), a gel was transferred onto a polyvinylidene difluoride membrane in 1× NuPAGE transfer buffer (in 20% methanol, wt/vol). The membrane was blocked with 5% nonfat dry milk (wt/vol) in buffer containing 1 M Tris-buffered saline and 0.1% Tween-20 (vol/vol) and probed with antibodies to GluA1 (1: 1000, Cell Signaling Technology, Danvers, MA, USA, #13185), and GluN1 (1: 1000, Cell Signaling Technology, #5704) at 4°C overnight. After rinses in TBS-Tween, the membrane was incubated for 1 h at 20–22°C in horseradish peroxidase–conjugated goat antibody to rabbit IgG (1: 3000, Fisher Scientific, #31462). The immunoblot was developed with enhanced chemiluminescence (Fisher Scientific). Levels of expression were computed using ImageJ (NIH). For illustration purposes, blots were cropped and the brightness and contrast were adjusted globally using Photoshop (www.adobe.com).