Protease inhibitor cocktail for mammalian cells and tissue extracts

After treatments cells were rinsed twice with ice-cold PBS and 300 µL precooled lysis buffer (CelLytic™ M, Sigma-Aldrich, St. Louis, MO, USA, or lysis buffer provided with the ELISA kit), supplemented with phosphatase and protease inhibitors (PhosSTOP™, Roche Diagnostics GmbH, Mannheim, Germany; Protease Inhibitor cocktail for mammalian cells and tissue extracts, P8340 from Sigma-Aldrich, St. Louis, MO, USA), were added to each well. Cellular proteins were extracted at 4 °C on a shaker for 15 min. Cell residues were scraped off the wells and the lysates were transferred to 1.5 mL precooled plastic tubes and centrifuged at 4 °C for 10 min at 18,000 g. The supernatants were transferred into fresh tubes, vortexed, and briefly sonicated. If needed, protein concentration in extracts was determined via BCA assay (Pierce^® BCA Protein Assay Kit) according to the protocol provided by the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA).

The brain tissues (∼0.4 g) were thawed on ice and homogenized using Ultra-turrax T8 homogenizer (IKA-Werke GmbH & Co. KG, Germany) in 500 µl solubilization buffer containing 8M urea, 2% (w/v) CHAPS, 0.2% sodium orthovanadate and 1× concentration of protease inhibitor cocktail for mammalian cells and tissue extracts (Sigma Aldrich, USA). The supernatant was collected by centrifugation and subjected to three pulses of sonication on ice followed by centrifugation at 20,000× g for 30 min at 4°C for the recovery of total soluble proteins. Protein extraction from Neuro2a cells was carried out in a similar manner. Protein concentration was quantified in supernatants by Bradford Protein Assay kit (Bio-Rad, USA) as per manufacturer's instructions and the extraction procedure was carried out according to [13] (link).