Ecl western blotting kit

Total protein was extracted from the frozen tissues using lysis buffer on ice. Bradford method was used to quantify the protein. Equal amounts of protein (20µg) were subjected to 12% polyacrylamide, 0.1% SDS (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, USA). Following blocking in phosphate-buffered saline with 0.1% Tween-20 (PBST) containing 0.5% skim milk powder for 1 hour at room temperature (RT), the membranes were incubated in different primary antibodies overnight at 4°C. The primary antibodies used were rabbit anti-CLDN8 antibody (1:10,000; Abcam, Cambridge, MA, USA) and mouse anti-GAPDH antibody (1:5000, Santa Cruz Biotechnology, CA, USA). After washing by PBST, the membranes were incubated with HRP-linked anti-rabbit or HRP-linked anti-mouse secondary antibodies for 1 hour at RT. The detection was carried out with an ECL Western blotting kit (BioVision, San Francisco, USA), the density of protein bands was analyzed by Adobe Illustrator CS6 analysis software.

Cells were lysed using RIPA buffer (50 mM Tris, pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 0.5% sodium deoxycholate and 150 mM NaCl), and the protein concentration was determined using the bicinchoninic acid assay (BCA) method. Briefly, 200 µl of the BCA working solution and 20 µl of diluted sample protein were added per well into 96-well plates and the absorbance was measured at 526 nm after incubation at 37°C for 30 min. Protein concentration was calculated in relation to the standard curve. Protein samples (50 µg) were then separated using 10% SDS-PAGE and transferred to PVDF membranes, followed by blocking at 25°C for 1 h in 5% non-fat milk powder. Membranes were then incubated at 4°C overnight with primary antibodies. The membranes were then washed with TBS-Tween buffer to remove the free protein and incubated with anti-goat and anti-rabbit IgG H&L (HRP) (cat. no. ab205718; dilution 1:5,000; Abcam) secondary antibodies at 25°C for 1 h. The protein bands were visualized using the ECL Western blotting kit (cat. no. WP20005; Thermo Fisher Scientific, Inc.), and the bands were exposed to autoradiography film. β-actin was used as the loading control. Each experiment was repeated three times.

Total proteins were extracted by using RIPA Lysis Buffer (cat. no. P0013E; Beyotime Institute of Biotechnology). The BCA Protein Assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology) was used to determine the concentration of protein extracted from each sample. SDS-PAGE (on 10% gel) was then adopted to isolate the protein (40 µg) from each sample after which samples were removed to a polyvinylidene fluoride (PVDF) membrane. The film was then blocked for 1 h in 5% nonfat milk at 4°C. The membrane was then incubated in primary monoclonal and secondary antibodies (all from Abcam) against GAPDH (cat. no. ab8245, dilution 1:1,000), pro-caspase-3 (cat. no. ab179517, dilution 1:1,000), cleaved caspase 3 (cat. no. ab2302, dilution 1:500), Bcl-2 (cat. no. ab32124, dilution 1:500), Bax (cat. no. ab205718, dilution 1:500) for 2 h at 4°C. The secondary antibodies were anti-rabbit (cat. no. ab205718, dilution 1:1,000) conjugated with horseradish peroxidase for 1 h at room temperature. Lastly, the signals were visualized using an enhanced ECL Western blotting kit (Abcam), and the optical density of each band was analyzed by Gel-Pro-Analyze software (version 4.5; Media Cybernetics, Inc.).