Caspase 8 Monoclonal Antibody (90A992)

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	-Mouse (1:0000)

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-Mouse (1:0000)

Publication protocol

Following treatment with BHP, the T24 cells were washed with PBS and lysed in ice-cold lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM sodium orthovandate and 1 mM NaF. The cell lysates were centrifuged at 12,000 × g for 45 min at 4°C to collect the supernatant. A Bradford protein assay was used to determine the protein concentration. The proteins (40 µg) were separated using 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked using 5% non-fat milk, followed by overnight incubation with primary antibodies at 4°C. The primary antibodies used were as follows: Cyclin E (1:1,000; cat. no. sc-377100), cell division cycle 25C (Cdc25c; 1:1,000; cat. no. sc-13138), p21 (1:1,00; cat. no. sc-6246), Bcl-2-associated X protein (Bax; 1:1,000; cat. no. sc-526), apoptosis-inducing factor (AIF; 1:1,000; cat. no. sc-5586), caspase-3 (1:1,000; cat. no. sc-7148), and poly(ADP-ribose) polymerase (PARP; 1:2,000; cat. no. sc-7150) all obtained from Santa Cruz Biotechnology, Inc., and phosphorylated (p)-p53 (1:50; cat. no. PA5-27822), B cell lymphoma-2 (Bcl-2; 1:200; cat. no. PA1-28275), caspase-9 (1:1,000; cat. no. KHZ0102) and caspase-8 (1:1,000; cat. no. MA1-41280) all obtained from Thermo Fisher Scientific, Inc., and endonuclease G (Endo G; 1:1,000; Koma Biotech, Seoul, Korea). Following incubation with primary antibodies, the membranes were washed with Tween and PBS and then incubated again for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:3,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA; cat. no. 170–6515). Electrochemiluminesence reagent (GE Healthcare Life Sciences, Amersham, UK) was used to analyze the bound antibody complexes and β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-47778) served as an internal control. A gel imaging analysis system (Kodak ID; Kodak, Rochester, NY, USA) was used to analyze the bands.

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