Phosphatase cocktail inhibitors 1 and 2

The following antibodies were used in this study: rabbit anti-GRP78 and rabbit anti-ERp29 from Novus Biologicals (Littleton, CO); rabbit anti-eIF2a, mouse anti-phospho-eIF2a (S51), rabbit anti-p58^IPK, rabbit anti-cleaved PARP, rabbit anti-spliced XBP-1 (XBP-1s), rabbit anti-cleaved caspase 3and mouse anti-GADD153/CHOP from Cell Signalling Technology (Beverly, MA); mouse anti-β-actin from Sigma-Aldrich (St Louis, MO); rabbit anti-CaMKII, rabbit anti-Bax, rabbit anti-caspase 12 and rabbit anti-phospho-CaMKII (T286) from Abcam (Cambridge, MA).
Fucoidan isolated from Fucus vesiculosus was purchased from Sigma-Aldrich (Saint Louis, MO). The complete, EDTA-free protease inhibitor cocktail tablets were obtained from Roche Diagnostics (Indianapolis, IN). Phosphatase cocktail inhibitors I and II and thapsigargin (TG) were from Sigma-Aldrich (Steinheim, Germany). Lipofectamine 2000 transfection reagents were supplied from Invitrogen (Eugene, OR). Salubrinal was purchased from Tocris Bioscence (Ellisville, MO).

Cells at 70–80% confluence were treated with fucoidan at various concentrations (0, 1.0, 5.0, 10, 50 and 100 µg/ml, respectively) for 3 days. Both the attached and suspended cells were harvested for protein extraction. Cell lysates were extracted with RIPA Buffer (1% Igepal, 1% sodium deoxycholate, 0.15 M sodium chloride, 0.01 M sodium sulphate, pH 7.2 and 2 mM EDTA), supplemented with protease inhibitors (Roche Diagnostics) and phosphatase cocktail inhibitors I and II (1∶100, Sigma-Aldrich). Western blotting was carried out as described [28] (link). Briefly, total proteins (30 µg/lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline buffer with 0.1% Tween 20 for 1 h at room temperature and probed with the indicated primary antibodies. Goat anti-mouse horseradish peroxidase (HRP; Upstate Biotechnology, Lake Placid, NY) or goat anti-rabbit HRP secondary antibody (ZyMED Laboratories, San Francisco, CA) was used as secondary antibodies. The chemiluminescent signal was developed with Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Signal intensity was analysed using GeneTools software (Syngene, Frederick, MD). The level of β-actin was used as a loading control.