Image quant imager

CCA cells were treated with various concentrations of the extracts for 48 hrs. After collecting the living cells, these were washed with ice-cold phosphate buffered saline and lysed using RIPA lysis buffer (0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 50 mM Tris, 1% Tween 20 and protease cocktail inhibitor). The protein concentration was determined using BCA Protein assay. Equal amounts of protein were resolved in 4x SDS buffer and boiled at 95°C. Then, proteins were loaded and separated by 10% (w/v) SDS-polyacrylamide gel electrophoresis before being transferred to a polyvinylidene fluoride membrane (Immobilon, Merck). After blocking the non-specific sites with 5% (w/v) skimmed milk in Tris-buffered saline (TBS), the membrane was probed with primary Ab, mouse anti-actin Ab (1:10,000), mouse-anti-BAX Ab (1:1,000) and rabbit-anti Bcl-2 Ab (1:1,000). Then, the membrane was washed with TBS containing 0.1% Tween 20 (TTBS) 3 times and TBS once before being incubated with secondary Ab conjugated with horseradish peroxidase. Immunodetection was performed using ECL. The apparent density of the bands on membranes was captured using ImageQuantTM Imager (GE Healthcare UK Ltd., UK). The experiments were performed in triplicate.

The whole cell lysates were prepared using RIPA lysis buffer. The 20 μg of total protein was electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Whatman, Dassel, Germany). The membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with primary antibodies against HIF-3α (1:500, Aviva systems biology, CA, USA), HIF-1α (1:200, Abcam, Cambridge, UK) and β-actin (1:10,000, Abcam, Cambridge, UK) at 4°C overnight. After incubation with a secondary antibody, the band intensity was measured using ECL^TM Prime Western Blotting Detection Reagent for chemiluminescence detection (GE Healthcare UK Ltd., UK). The apparent density of the bands on membranes was captured by ImageQuant^TM Imager (GE Healthcare UK Ltd., UK).

The human phospho-kinase array kit (ARY003B, R&D system, Minneapolis, MN) containing 46 phosphokinases printed in duplication was used to screen the signaling pathway. Briefly, the 80% confluent NG or HG cells were lysed with NP-40 lysis buffer containing phosphatase- and protease-cocktail inhibitors (Roche, Mannheim, Germany) and incubated at 4 °C for 15 min. Cell lysate was obtained after centrifugation at 12,000 xg, 4 °C, for 15 min and the total protein was quantified using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA) according to the instructions of the manufacturer. Total protein lysate (600 μg) was incubated with an antibody-array membrane at 4 °C, overnight. The membrane was then incubated with cocktail-detection antibody and streptavidin horseradish peroxidase. The signals were detected by Chemireagent provided in the same kit and quantified using a Image Quant™ Imager (GE Healthcare Bioscience AB, Uppsala, Sweden).