Mf chemibis 3.2 imaging system

The cells were washed with ice‐cold PBS and lysed in a modified RIPA buffer containing 1 mM DTT, 1 mM PMSF, complete protease inhibitor cocktail (1 tablet/10 mL; Invitrogen, Carlsbad, CA, USA). The cell lysate was sonicated and followed by centrifugation at 12 000 g for 20 minutes at 4°C. Protein concentration was measured with BCA protein assay kit (Beyotime Biotechnology). Western blots were performed with specific antibodies to detect the corresponding proteins. After incubation at 4°C overnight, the blot was washed three times with 0.05% Tween‐20 TBS (TBST), and then incubated with 1:10 000 diluted goat anti‐rabbit/anti‐mouse IgG conjugated with HRP for 2 hours at room temperature. After additional washing with TBST, the target proteins on the blot membrane were visualized using the ECL system. The MF‐ChemiBIS 3.2 Imaging System (DNR Bio‐Imaging Systems, Jerusalem, Israel) was used for image capture. To control sampling error, the same blot was also probed for β‐Actin or GAPDH as an internal loading control. The integral optical density of each band was analysed using the Image‐J software and the ratio of band intensities of target protein over associated control was obtained as the statistic value. Data were expressed as the mean ± SD of at least three independent experiments.

Cells were centrifuged at 250×g for 5 min and lysed in 200 µl RIPA buffer (Abcam, Cambridge, UK) supplemented with 20 µl/ml protease and phosphatase (Serva Electrophoresis, Heidelberg, Germany) inhibitor cocktails. Sample volumes equivalent to 50 μg of protein were prepared in Laemmli SDS sample buffer (Thermo Fisher Scientific) and incubated at 85 °C for 2 min. 20 µl sample per lane were separated by standard SDS-PAGE on 4–12% precast gels (Serva) and electrophoretically transferred to PVDF membranes (Thermo Scientific). After 1 h blocking in TBS (pH 7.6) containing 5% BSA and 0.1% Tween-20, the membranes were incubated overnight at 4 °C with mouse anti-p53 (Santa Cruz Biotechnology, Heidelberg, #sc-126, RRID: AB_628082; 1:100) antibody. Equal loading of protein was verified by using rabbit anti-GAPDH (Cell Signaling, Danvers, MA, USA, #2118, RRID: AB_561053; 1:3000). HRP-conjugated anti-mouse IgG (Cell Signaling, #7076, RRID: AB_330924; 1:3000) and HRP-conjugated anti-rabbit IgG (Cell Signaling, #7074, RRID: AB_2099233; 1:20,000) were used as secondary antibodies followed by detection of specific signals using Immobilon Forte Western HRP Substrate (Sigma Aldrich). Imaging was done on an MF ChemiBis 3.2 imaging system (DNR Bio Imaging Systems, Neve Yamin, Israel).

Total protein lysates were isolated from treated confluent CAOV3 cells using lysis buffer (0.01 mmol/l Tris-HCl, pH 7.6, 0.1 mmol/l NaCl, 0.001 mol/l EDTA, pH 8.0, 1 μg/ml Aprotinin, 100 μg/ml PMSF). Protein concentrations were determined by BCA Protein Assay Kit (PIERCE, Rockford, IL). Proteins were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes, which were then blocked for 2 h in 5% defatted milk in Tris-buffered saline containing Tween-20 (TBST, 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20). Membranes were then incubated for 2 h at room temperature with the following primary antibodies: ADM (1:1000, Phoenix, USA), HIF-1α (1:200, Transduction Laboratories, Heidelberg, Germany), VEGF (1:200, Phoenix, USA) and GAPDH (1:1000 Keygen Biotech, China). Primary antibodies were recognized by appropriate HRP-conjugated secondary antibodies (GE Healthcare Life Sciences, Piscataway, NJ) and visualized using the enhanced chemiluminescence detection system (ECL, Beyotime Institute of Biotechnology, China). The MF-ChemiBIS 3.2 Imaging System (DNR Bio-Imaging Systems, Israel) was used for image capture.