D-MEM, FBS, and tissue culture reagents were obtained from Life Technologies. The anti-GRP 78, CHOP and actin antibodies were supplied by Santacruz (Santa Cruz, Calif., USA). All other chemicals were purchased from Sigma. All reagents were at least of analytical grade.
Preparation of BS extracts
Hot water-extracted BS was prepared and concentrated to 1 g of medicine per ml of distilled water. After dissolution and mixing in PBS at 37°C for 30 min, the concentration was adjusted to 100 mg/ml. This solution was then centrifuged (1200 g, 30 min) to remove any insoluble ingredients. The supernatant was sequentially passed through 0.45 μm filters for sterilization. The extracts were used in all experiments.
The clonal human cervical carcinoma HeLa cells were obtained from ATCC. The cells were maintained in D-MEM supplemented with 10% FBS, penicillin G (100 U/ml), and streptomycin (100 μg/ml) in a humidified incubator.
Measurement of calpain activity
The calpain activity was measured in the cytosolic fraction using a calpain assay kit purchased from Calbiochem (San Diego, Calif., USA). The assay is based on the fluorometric detection of the cleavage of the calpain substrate, Suc-Leu-Tyr-7-amino-4-methylcoumarin (Suc-Leu-Tyr-AMC, 50 μM). The reaction buffer (145 mM NaCl, 100 mM Tris-HCl, pH 7.3) and substrate were then added to all samples. The reaction was carried out in a 96-well plate that was protected from light and incubated for 1 h at 37°C with constant shaking. The samples were read in a Gemini fluorescence microplate reader (Molecular Devices) using 360 nm excitation and 430 nm emission filters. All measurements were performed in triplicate, and repeated with heart extracts in two independent experiments.
Western blot analysis
Western blot analysis was performed as described previously [7
]. Briefly, whole-cell lysates were generated using a buffer consisting of 1% Nonidet P-40, 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 10 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 100 mM sodium fluoride. Equal amounts of the lysates were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore) in a transfer buffer [25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol]. The membranes were first rinsed in Tris-buffered saline (TBS: 10 mM Tris [pH 7.4], 150 mM NaCl) and then blocked overnight at room temperature in TBS-5% bovine serum albumin (BSA). Various antibodies including the anti-Bax antibody were each used at a dilution of 1:1,000 in TBS-5% BSA. The antibody–antigen complexes were detected using horseradish peroxidase-conjugated protein A or horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad) and a chemiluminescent substrate development kit (Kirkegaard and Perry Laboratories). An equal loading was determined by the presence of β-actin.
The data was analyzed by one-way analyses of variance using Microcal Origin software. The differences between the treated samples were analyzed using the individual contrast when the factor consisted of more than two levels. P values < 0.05 were considered significant.