Ecl kit

Total protein was extracted from CHON-001 cells and tissues with cell extraction kit (Thermo Scientific, Waltham, MA, USA) by centrifuging samples at 4°C, 6,000 × g for 10 min. A BCA kit was used to determine the concentration of extracted protein. Total protein (20 µg) and a pre-stained protein ladder (Thermo Fisher Scientific, Inc.) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Sigma-Aldrich; Merck KGaA). Membranes were stained with 1X ponceau-S (Beijing Solarbio Science & Technology, Co. Ltd.) following transfer to ensure consistent loading of total protein per lane. The protein membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature. Primary antibodies (Table II; Cell Signaling Technologies, Inc.) were applied and membranes were incubated at 4°C overnight. Secondary antibodies (cat. nos. 7074 and 7076; Cell Signaling Technologies, Inc.) were applied for 2 h at room temperature. Primary and secondary antibodies were diluted with TBST with Tween-20, as specified by the supplier. An ECL kit (Sigma-Aldrich; Merck KGaA) was employed to visualize proteins. Stains were developed with X-ray film (Fuji, Tokyo, Japan). The densitometry was performed using the Bio-Rad ChemiDoc system with Image Lab software version 6.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A549 cells were seeded in 6-well plates for 12 h, and then treated with either homoisoflavanone-1 (12.5, 25, 50, or 100 µg/ml) or DMSO for 24 h. Cellular proteins were harvested and immunoblotting was carried out as previously described (21 (link)) with appropriate modifications. Cells were resuspended in lysis buffer (1% Triton-X100, 50 mM Hepes pH 7.4, 2 mM sodium orthovanadate, 1 mM edetic acid, 100 mM sodium fluoride, 1 mM PMSF, 10 µg/ml of leupeptin and 10 µg/ml of aprotinin), and the supernatants of the lysates were collected after centrifugation at 14,000 g for 10 min at 4°C. The protein concentration was determined using a protein concentration kit. Proteins were separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyldene fluoride membranes. The membranes were blocked in 5% skimmed milk for 2 h at room temperature. Protein was detected with antibodies against active caspase 3, caspase 3, PARP, Bcl-2, Bak, p-Cdc2, Cdc2, p-P38, P38, P53 or β-actin at 4°C. The membranes were washed three times with TBST before incubation with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Immunoreactive bands were visualized using an ECL kit (Sigma-Aldrich; Merck KGaA). Protein levels were quantified relative to the control group.

The protein we used was extracted by utilizing RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA, USA). And Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was used for assessing the content of the proteins. Loaded equal amounts of proteins on SDS-GAGE and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). Next, the membranes were blocked with 5% non-fat milk for 1 h, and incubated with applied primary antibody (dilution, 1:1000) overnight at 4°C, after that, using HRP-conjugated secondary antibodies (dilution, 1:1000) to incubate for 1 h. Subsequently, the membranes were visualized by using ECL kit (Sigma). After all, corresponding internal parameters were analyzed by Image Lab^TM Software (Bio-Rad Laboratories). Uncropped figures of the Western Blot Assay were shown in Supplementary figures S1-7.