Nitrocellulose filter nc membrane

The GLS expressions were detected by Western blot analysis. After treatments, cells were harvested and lysed by RIPA buffer containing protease inhibitor cocktail (Sigma, St. Louis, MO, U.S.A.). Lysates were incubated at 4°C for 30 min followed by centrifugation at 12000 rpm for 10 min at 4°C. The protein concentrations were measured by Bradford assay (Beyotime, China). Protein samples (30 μg) were separated by 10% SDS/PAGE. Proteins were transferred on to 0.22 μm-pore-diameter nitrocellulose filter (NC) membranes (Millipore, U.S.A.). Membranes were blocked with 5% BSA for 1 h at room temperature, followed by incubation with primary rabbit antibodies against GLS (ab93434, Abcam, U.S.A.) (1:1000) and β-actin (Boster Bio, China) (1:2000) for overnight at 4°C. After washing with TBS/T three times, the membranes were incubated with secondary antibody (1:3000) for 1 h at room temperature. Then, the membranes were washed and scanned by using an Odyssey Infrared Imaging System (LI-COR, U.S.A.).

After treatments, cells were harvested and lysed using RIPA lysis buffer (Beyotime, Biotechnology, China) containing protease inhibitor cocktail (Roche). Protein concentrations were detected using the BCA method, and equal amounts of protein for each lysate sample were subject to SDS-PAGE at 80–130 V for 90–120 min, and then transferred to nitrocellulose filter (NC) membrane (Millipore, Boston, MA, USA). After blockade in tris-buffered saline and Tween-20 (TBS-T; 10 mM Tris, 150 mM NaCl, and 0.1% Tween-20) containing 5% nonfat dry milk or 3% bovine serum albumin (BSA), the membranes were incubated with indicated primary antibodies diluted in BSA buffer, overnight at 4 °C. The membranes were washed three times in TBS-T followed by 1 h incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) at room temperature. Then the membranes were washed before enhanced chemiluminescence exposure. GAPDH or β-actin were used as housekeeping controls. Chemiluminescent detection was carried out on the ChemiDoc™ XRS + System with Image Lab™ Software (Bio-Rad, Hercules, USA). Antibody information was showed in Table S1.
For cytokine concentration measurements, ELISA was performed according to manufacturer’s instructions using cell supernatants following incubation and centrifugation. The ELISA kits are listed in Table S1.

RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Beyotime, Shanghai, China) was used to separate total protein from human glioma cells and tissues. The concentration of total protein was determined with a BCA kit (Solarbio, Beijing, China). An equal amount of total protein (30 µg) was electrophoresed on 10%−12% SDS-PAGE, and then electro-transferred to a nitrocellulose filter (NC) membrane (Millipore, MA, USA). Then, the membranes were blocked with 5% non-fat dry milk in TBST at RT for 1 h. After washing with TBST, the membranes were incubated with subsequent primary antibodies at 4 °C overnight. On the second day, the membranes were incubated with HRP-labeled secondary antibodies (Proteintech, Wuhan, China) at RT for 1 h. An enhanced chemiluminescence system (Millipore, MA, USA) was used to measure protein expression value. The relative quantity of proteins was analyzed by Image J software. The antibodies are shown in Table S2.

Cells were collected and lysed in protein lysis buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM Ethylene Diamine Tetraacetic Acid (EDTA), 1% (m/v) Nonidet P-40 (NP-40), 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mM NaF and 1.0 mM dithiothreitol). The lysates were clarified by centrifugation at 4 °C for 20 min at 14,000 rpm. The concentration of protein in the supernatants was measured using a BCA assay kit. An equal amount of protein lysate (50 μg) was placed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for 2 h at 100 V, followed by transfer of the protein to an Nitrocellulose filter (NC) membrane (Millipore, Boston, MA, USA). The blots were blocked with 3% BSA in PBS at 37 °C for 1 h and were incubated with specific antibodies against indicated primary antibodies overnight at 4 °C. The membranes were washed three times with PBS containing 1% Tween-20, and probed with IRDyeTM 800-conjugated secondary antibody for 1 h at 37 °C. Blots were reacted with enhanced chemiluminescence (ECL) reagent (Bio-Rad, Hercules, CA, USA) and signals were detected by Amersham Imager 600 (GE, Piscataway, NJ, USA).