Ecl detection system

Western blot analyses were performed as previously described [33 (link)]. Briefly, cells were washed twice with PBS and then lysed via sonication in lysis buffer (Intron, Seoul, Korea). The samples were separated on 10–15% SDS-PAGE gels, and then transferred to nitrocellulose membranes (Protran BA83; Whatman). Immunoblotting was performed using the following primary antibodies and dilutions: anti-Capase 8 (1:1000; Cell Signaling, Danvers, MA, USA), anti-Caspase 3 (1:1000; Cell Signaling, Danvers, MA, USA), anti-Caspase 7 (1:1000; Cell Signaling, Danvers, MA, USA), anti-cleaved Caspase 7 (1:1000; Cell Signaling, Danvers, MA, USA), PARP (1:1000; Cell Signaling, Danvers, MA, USA), BCL2 (1:1000; Cell Signaling, Danvers, MA, USA), DR4 (1:1000; Abcam, Cambridge, MA), DR5 (1:000; Abcam, Cambridge, MA, USA) and anti-beta actin (1:10000; Sigma, St. Louis, MO, USA). Horseradish peroxidase-labeled rabbit anti-mouse (Abcam, diluted 1:5000, Cambridge, MA, USA) and goat anti-rabbit (Santa Cruz, diluted 1:2000, Finnell Street, Dallas, TX, USA) secondary antibodies were used. The proteins were visualized using an ECL detection system (Ab Frontier, Seoul, Korea).

After experimental treatments, Western blotting was performed as previously described [12 (link)]. Whole-cell lysates and tissue lysates were obtained by using an EzRIPA Lysis kit (ATTO, Tokyo, Japan; 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, as well as protease inhibitor and phosphatase inhibitor cocktails). Protein concentrations in lysates were determined by using a BioRad protein assay kit (BioRad Laboratories, Hercules, CA, USA). SDS-PAGE was performed with the protein samples, after which the proteins were transferred to a nitrocellulose membrane. For blocking, 5% bovine serum albumin in TBST (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20) was used for 1 h at room temperature. Membranes were then incubated at 4 °C overnight with specific primary antibodies and subsequently probed with peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 h at room temperature. Blots were visualized by using an ECL detection system (Abfrontier, Seoul, Republic of Korea). Data acquisition and densitometric analysis were performed using an iBright CL1000 imaging system (Thermo Fisher Scientific, Waltham, MA, USA).