Supersignal west pico chemiluminescence substrate

Sphingo strips (Echelon Biosciences Inc., Salt Lake City, UT) were probed with various proteins according to the manufacturer’s instructions. Briefly, the Sphingo strips were blocked at RT for 2 h in blocking buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween-20 and 3% BSA, pH 8.0) and washed three times with wash buffer (10 mM Tris, 150 mM NaCl and 0.1% Tween-20, pH 8.0). Purified BmTFF3 was incubated with the Sphingo strips at RT for 1 h in blocking buffer at a concentration of 2 μg ml⁻¹. Then, the protein solution was removed and washed three times with wash buffer. A rabbit anti-BmTFF3 polyclonal antibody (1:1000 dilution) was used as the primary antibody, and HRP-conjugated goat anti rabbit secondary antibodies (1:5000 dilution) were used as the secondary antibody. Binding was detected with the SuperSignal WestPico chemiluminescence substrate (Invitrogen).

Western blotting was performed for the detection of βγ-CAT oligomerization as described previously^{49 (link)}. The cells were treated with 5 nM βγ-CAT or a βγ-CAT/GSL mixture for 1 h, then the cells were lysed and separated by 12% SDS−PAGE and then electrotransferred onto polyvinylidene difluoride membranes. The membranes were subsequently blocked with 3% BSA and sequentially incubated with a rabbit anti-βγ-CAT polyclonal antibody (1:1000 dilution) and HRP-conjugated goat anti-rabbit secondary antibodies (1:5000 dilution). Finally, the protein bands were visualized with the SuperSignal WestPico chemiluminescence substrate (Invitrogen).
To detect the expression of caspase p20 and mature IL-1β in frog peritoneal cells, frog peritoneal cells were first primed with LPS (100 ng ml⁻¹) for 2 h at RT and then incubated with βγ-CAT (100 nM) for 1 h at RT. Finally, the peritoneal cells were lysed for the detection of caspase p20 or βγ-CAT oligomerization by western blot, and the supernatants were concentrated to 1/10th of the original volume for the western blot analysis of mature IL-1β expression.

Worms were collected in maintenance (M9) buffer and washed several times until no visible bacterial contamination was observed in the washing M9 buffer. Worm pellets were froozen overnight at -80°C, and pellets were lysed with buffer containing 150 mM NaCl, 50 mM Tris:HCl, pH 7.4, 1% (v/v) Triton X-100, 0.1% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) sodium deoxycholate (w/v), 10 μg/ml leupeptin, 10 μg/ml chymostatin and 10 μg/ml pepstatin A. Pellets were directed through 271/2G syringes (10-fold), sonicated and centrifuged at 16,000g. Protein in supernatants were collected and their concentrations measured by bicinchoninic acid assays as described in the manufacturer's protocol (Thermo Fisher Scientific). Bcl-xL(54H6) rabbit monoclonal Ab (Cell Signaling Technology, Whitby, ON), and actin (AC-15) mouse monoclonal Ab (Abcam Inc., Cambridge, MA) were tested in this study. Peroxidase-labeled secondary Ab were detected by enhanced chemiluminescence with reagent set from GE Healthcare Life Science (Mississauga, ON) or SuperSignal WestPico chemiluminescence substrates (Thermo Scientific, Rockford, IL). Densitometry analysis, were analysed by Image J software (v1.49), a Java-based processing program developed by the National Institutes of Health (USA).