Western Blot Analysis of Apoptosis Markers

The preparation of SCC-9 and HSC-3 cell lysates for the Western blot analysis followed previously described procedures [18 (link)]. The Western blot analysis was performed with indicated primary and horseradish peroxidase-conjugated secondary antibodies. The primary antibodies were used as follows: pro-caspase-8 (#9746), pro-caspase-9 (#9502), cleaved caspase-8 (#9496), cleaved caspase-9 (#9505), cleaved caspase-3 (#9664), PARP (#9542), p-ERK (#4370), ERK (#9102), p-JNK (#4668), JNK (#9258), cIAP-1 (#7065, Cell Signaling Technology, MA, USA); pro-caspase-3 (610323), p38 (612168), p-p38 (612281, BD biosciences); HO-1 (ab68477), beta-actin (ab8226, Cambridge, MA, USA). Then, HRP-conjugated anti-mouse IgG (5450-0011, Seracare Life Sciences, Milford, MA, USA) or anti-rabbit IgG (5450-0010, Seracare Life Sciences, MA, USA) secondary antibodies were applied. All the blots were carried out with an enhanced chemiluminescence substrate solution (EMD Millipore Corporation, Burling, MA, USA) to produce images. The band intensities were quantified by NIH ImageJ analysis software.