Pmsf serine protease inhibitor

Snap-frozen V1 tissues were homogenized in either TRIzol LS Reagent (ThermoFisher), or NP40 (ThermoFisher) lysis buffer, and protein extracted according to manufacturer’s instructions. Subsequent lysates were supplemented with 10% Protease Inhibitor Cocktail (PrIC; Sigma-Aldrich), 1% Phosphatase Inhibitor Cocktail 2 (PhIC; Sigma-Aldrich), 1 mM PMSF Serine Protease Inhibitor (Sigma-Aldrich). Protein concentration was determined using Bradford Reagent (Sigma-Aldrich). At experimental endpoints, cells were lysed using RIPA buffer (ThermoFisher) and addition of previously mentioned inhibitor cocktails. Marmoset and human frontal cortical tissues were also lysed in this way for use as positive controls. Equal concentrations of samples were added to 4X Loading Buffer (240 nM TRIS, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol, 0.05% bromophenol blue), heated at 95 °C for 10 min, and electrophoretically separated on a 4–12% Bis-Tris gel (ThermoFisher). Following gel electrophoresis, proteins were blotted onto PVDF membranes (ThermoFisher), pre-blocked in Odyssey Blocking Buffer (PBS; Li-Cor) before incubation with primary antibodies (Supplementary Table 1) overnight at 4 °C. Following washes, membranes were incubated with IRDYE secondary antibodies (Li-Cor) and visualized using the Odyssey CLx Scanner (Li-Cor).

Total cell lysates were prepared using lysis buffer (Biosource International) supplemented with Phosphatase Inhibitor Cocktail 2 (P5726, Sigma Aldrich), Phosphatase Inhibitor Cocktail 3 (P044, Sigma Aldrich), Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF serine protease inhibitor (Sigma-Aldrich), and 10 µg of proteins were separated by SDS-PAGE using Bolt 4–12% Bis–Tris Plus gels (Invitrogen). Gels were transferred onto nitrocellulose membranes using Power Blotter Select Transfer Stacks and the Power Blotter System (Invitrogen). Membranes were blocked with I-Block reagent (ThermoFisher) and incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal anti-calnexin (sc-23954, Santa Cruz); mouse monoclonal anti-CD63 (ab213090, abcam); mouse monoclonal anti-CD81 (ab59477, abcam); mouse monoclonal anti-GAPDH (GTX627408); mouse monoclonal anti-Cytochrome C (SC-13156); mouse monoclonal anti-HSP70/HSC70 (W27, SC-24). Membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody, Goat anti-Mouse (G-21040). Images were acquired using Westar Hypernova ECL substrate (Cyanagen) and iBright Western Blot Imaging Systems (Invitrogen).