Las 3000 image analyzer

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.

Western blot analyses were performed utilizing standard procedures. Briefly, 35 μg of isolated protein from cell lysates were loaded and resolved using standard 7.5 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore, Billerica, MA, USA) by electroblotting, which were blocked with fat-free milk and probed with specific antibodies against E-cadherin (1:1000, BD Biosciences, San Diego, CA, USA) and fibronectin (1:5000, abcam). Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection reagents (GE Healthcare, Bloomington, IL, USA), and then digitized using the LAS 3000 image analyzer. Tubulin (1:5000, Sigma, St. Louise, MO, USA) was used as loading control. Quantitative changes in band intensities were evaluated using Image J software (version 1.50; National Institute of Health, Maryland, WA, USA).