Quantitative Analysis of CED-9 Protein

A total of 300 synchronized wild-type N2 worms at day 1 and day 4 adult stages supplemented with DMSO or DIM for 24 h were harvested in 10 μL of protease inhibitor cocktail per experiment and lysed at 100 °C for 10 min. Each sample (18 μL) was loaded onto a 4–12% SDS-PAGE gel (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membrane (PROTRAN BA83, Whatman, Sigma-Aldrich, St. Louis, MO, USA), and the procedure was followed as previously described [22 (link)]. Primary antibodies used were rabbit anti-CED-9 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and mouse anti-α-tubulin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and HRP-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA). Blots were visualized with an ECL western blot detection kit (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA), detected using the LAS-3000 image analyzer, and quantified using ImageJ software.