Proteases and phosphatases inhibitors

After deep anesthesia with carbon dioxide, the brain was quickly collected, washed with ice‐cold PBS, and dissected. For immunohistochemistry analysis, half brain was fixed 48 hr in PBS + 4% paraformaldehyde and embedded in paraffin. Whole hippocampal and cortical lysates were prepared by homogenizing frozen tissue in T‐Per extraction buffer (150 mg/ml, Pierce), complemented with proteases and phosphatases inhibitors (Fisher Scientific). Lysates were centrifuged at 20,000 × g for 30 min at 4ºC.
For crude synaptosomes, the hippocampus was dissected, placed in 1.5 ml 320 mM sucrose + 10 mM HEPES (pH 7.4) complemented proteases and phosphatases inhibitors (Fisher Scientific), and frozen at a controlled rate (−1°C/min) in CoolCell containers (Corning) in a −80°C freezer until further processing. Tissue was homogenized by hand using a glass–teflon grinder. Homogenates were centrifuged at 1,200×g for 10 min. The supernatant was further centrifuged at 12,500 × g for 20 min. The resulting pellet was resuspended in T‐Per complemented with proteases and phosphatases inhibitors and centrifuged at 1,200xg for 10 min (Figure S3a). All centrifugations were carried out at 4ºC. Protein concentrations were determined using a commercial Bradford assay (Bio‐Rad).

Using complete radio-immunoprecipitation assay lysis buffer (Santa-Cruz Technology, Santa Cruz, USA), with additional proteases and phosphatases inhibitors (Thermo Fisher, Aalst, Belgium), vessels were boiled at 95°C for 10 minutes. The partially lysed vessels were transferred to a BeadBeater (Biospec Products, Barlesville, USA) and beated for 2x30 seconds and cooled on ice in between. Protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, USA) and mixed with 4x sample loading buffer (BioRad, Veenendaal, The Netherlands), 20x reducing agent (BioRad) before boiling for 5 minutes at 95°C. Samples were cooled down to RT, centrifuged for 1 minute at 13,400 g and loaded on a gel (4–12% Bis-Tris, BioRad). The gel was blotted on a polyvinylidene difluoride membrane (BioRad) overnight 30V at 4°C. The blotting membrane was blocked by incubation with 3% milk powder in TBS-Tween and incubated with a primary antibody against sGC or β-actin (Abcam, Cambridge, UK and Sigma). Next, the blot was incubated with a peroxidase conjugated secondary antibody and bands were detected using the SuperSignal West Femto substrate (Pierce, Thermo Scientific, Rockford, IL, USA). Visualization was performed using the ChemiDoc XRS system (BioRad).