Proteinase inhibitor mixture

Colorectal tumor tissues (75–100 mg/mouse) from control and experimental mice were homogenized with 0.5 mL of ice-cold RIPA lysis buffer containing a proteinase inhibitor mixture (Roche) on ice for 30 min. The homogenate was rotated at 4°C for 20 min and centrifuged at 12,000/g at 4°C for 15 min and the supernatant was collected. Crude protein extracts (40 μg), the concentration of which were measured by BCA method, were denatured and fractionated by SDS-PAGE, electrotransferred onto nitrocellulose membranes (Millipore). The membranes were blotted in TBST (25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20) containing 5% nonfat milk or 5% BSA for 1 hour at room temperature and visualized using antibody against VEGF, COX-2, cyclin D1, survivin, and β-actin (1 : 1000, 1 : 500, 1 : 500, 1 : 500, and 1 : 2000, resp.) and detected by an enhanced chemiluminescence (ECL) detection system (Pierce). The density of the selected bands was quantified using ImageJ software.

Cells were washed with ice-cold PBS and incubated with RIPA buffer (Beyotime Institute Biotechnology, Shanghai, China) containing a proteinase inhibitor mixture (Roche, Mannheim, Germany) and 10 μM PMSF (Sigma, St. Louis, MO, USA). Lysates were briefly sonicated and cleared by centrifugation at 10,000 rpm for 20 min. Equivalent amounts of protein were analyzed using 10% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies. The following primary antibodies were used: anti-MAP2 (1:1000; Millipore Bioscience Research Reagents, Billerica, MA, USA), anti-PSD95 (postsynaptic density protein 95) (1:2000; Abcam, Cambridge, UK), anti-GluR1 (Glutamate Receptor 1) (1:300; Abcam), anti-MAOB (1:1000; Abcam), anti-GFAP (1:1000; Abcam), anti-GABA-T (1:1000; Abcam), anti-glutaminase (1:1000, Abcam), anti-β-actin (1:10000, Sigma). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse antibodies (1:10000, Boster Bio-Technology, Wuhan, China). Visualization was performed using enhanced chemiluminescence (ECL, GE Healthcare Pharmacia). Densitometric analysis of Western blots was conducted using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) and quantified using Quantity One version 4.1.0 (Bio-Rad).

Ischemic hemispheric brain tissues was homogenized in ice-cold RIPA lysis buffer (Merck Millipore) and proteinase inhibitor mixture (Roche Diagnostics). After clearing debris by centrifugation at 14,000 × g, protein concentration was determined using the BCA protein assay (Thermo Scientific, MA, USA). The lysate (500 μg) was incubated with anti-NR2B (2 μg; Millipore) or anti-tPA (2 μg; Molecular Innovations Inc.) antibody or normal IgG (2 μg, negative control) on a rotator at overnight 4 °C. Then, 20 μl Protein A agarose beads (Roche Diagnostics) were added and reacted for 6 hours at 4 °C. After washing with lysis buffer 3 times, the precipitates were used for Western blot.

RAW264.7 cells or macrophages from C57BL/6, TLR2^−/− or TLR4^−/− mice were stimulated with PPE26 (10μg/ml) for indicated time, and lysed with cell lysis buffer supplemented with a proteinase inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN, USA). Cell pellets were processed using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA) following the manufacturer's instructions. Equal amounts of proteins were separated on 10% SDS-PAGE and then transferred electrophoretically to PVDF membranes from Millipore (MA, USA). After treated with blocking buffer, the membranes were incubated with primary Abs overnight at 4°C, including rabbit anti-ERK2, rabbit anti-p38, rabbit anti-JNK, rabbit anti-phospho-ERK1/2, rabbit anti-phospho-p38, rabbit anti-phospho-JNK, rabbit anti-phospho-IκB-α, rabbit anti-NF-κB p65, rabbit anti-PCNA or rabbit anti-β-actin (Santa Cruz, CA, USA). After washed with TBST buffer, the membranes were incubated with the HRP-conjugated secondary Abs for 2 h at room temperature. Target proteins were visualized using the Pierce ECL Western Blotting Substrate (Pierce, Rockford, IL, USA).