Western Blot Analysis of Apoptosis Regulators

Western blot analysis was performed as described previously [28 (link)]. Whole animal protein extracts were obtained from 300 gravid adult hermaphrodites of each condition per gel well. Antibodies bound to a nitrocellulose membrane (PROTRAN BA83, Whatman, Sigma-Aldrich, St. Louis, MO, USA) were visualized with an ECL Western blot detection kit (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA), and the band intensities were measured with the LAS-3000 image analyzer using Multi Gauge software (v.3.0, Fuji Film, Tokyo, Japan). The following primary antibodies were used: rabbit anti-CED-9, a member of the anti-apoptotic Bcl-2 gene family in C. elegans (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-CED-4, the pro-apoptotic Apaf-1–like cell-death activator (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). The mouse anti-α-tubulin was used as the loading control (1:1000; Sigma-Aldrich, St. Louis, MO, USA). The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), HRP-conjugated donkey anti-goat IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and HRP-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA).