Bca protein measurement kit

The hippocampal tissues were homogenized in 500 μL ice-cold lysates. After centrifugation (12,000 r/min, 4°C, 10 min), protein concentrations were determined by the BCA protein measurement kit (Thermo Scientific, United States). Proteins were separated on 10% polyacrylamide electrophoresis gels (SDS-PAGE), then transferred onto PVDF membranes. Membranes were blocked with 5% skim milk powder in 1 × PBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) for 1 h, followed by incubation with rabbit-anti SPP1 (22952-1-AP, 1:3,000, Proteintech, United States), rabbit-anti SNCA (10842-1-AP, 1:1,000, Proteintech, United States), rabbit-anti SYN1 (20258-1-AP, 1:5,000, Proteintech, United States) and rabbit-anti GAPDH (10494-1-AP, 1:5,000, Proteintech, United States) at 4°C overnight. Membranes were washed with 1 × PBST three times for 15 min each after incubated. Then, membranes were incubated with horseradish peroxidase (HRP)-conjugated IgG of goat anti-rabbit (SA00001-2, 1:6,000, Proteintech, United States) for 90 min at room temperature, and washed with 1 × PBST three times for 15 min each. Subsequently, blots were detected by the ECL reagent (Thermo Scientific, United States). The protein bands were quantified using Quantity one v 4.6.2 software finally. GAPDH was used as a reference for relative expression.

The cultured cells was washed once with ice-cold PBS and lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris pH 8.0, 0.5% (v/v) DOC, 1% (v/v) NP40, 10% (v/v) glycerol, 1 mM aprotinin, 1 mM Pefabloc and 2 mM sodium orthovanadate). The tumor tissue from mouse experiments was lysed in the above lysis buffer with Tissuelyser II (Qiagen, Germany). After centrifugation, the supernatants were collected and protein concentrations were determined by BCA protein measurement kit (Thermo Scientific). Equal amount of protein from total cell lysate was run in SDS-PAGE and transfered on to polyvinylidine difluoride membranes. The membranes were blocked for 1 h with 5% BSA in TBS-T and then incubated with specific antibodies overnight at 4 °C. Then the membranes were washed 3 times with TBS-T, incubated for 1 h with a secondary horseradish peroxidase-conjugated antibody and developed with ECLWestern blotting detection reagent and detected with Amersham Imager RBG system (GE Healthcare, Buckinghamshire, UK).