Sc 134306

Pancreas and lung sections were incubated with the following primary antibodies: Anti-NRF2 (sc-365949, 1:200, Santa Cruz Biotechnology, CA, USA); anti-HO-1 (sc-136960, 1:200, Santa Cruz Biotechnology, CA, USA); anti-Mn-SOD (sc-137254, 1:200, Santa Cruz Biotechnology, CA, USA); anti-NLRP3 (sc-134306, 1:200, Santa Cruz Biotechnology, CA, USA); anti-Caspase-1 (sc-56036, 1:200, Santa Cruz Biotechnology, CA, USA); and anti-ASC (sc-514414, 1:200, Santa Cruz Biotechnology, CA, USA), as previously described [48 (link)]. Sections were then incubated with the following secondary antibodies: Peroxidase-conjugated bovine anti-mouse immunoglobulin G (IgG) or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA). Specific marking was revealed with a biotin-conjugated goat anti-rabbit IgG or biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Graphic presentation of densitometric analyses was performed Image J software (v1.52a) as previously described [49 (link)]. All immunohistochemical analyses were conducted by an observer without knowledge of the treatments.

The lysates of HL-1 (50 µg) underwent electrophoresis and were moved to a polyvinylidenfluoride (PVDF) membrane. Successively, the membranes were blocked in 5% non-fat milk in PBS 0.1% Tween-20, and then the blotted membranes were incubated overnight at 4 °C with the following primary antibodies: anti-TLR4 (1:500) (sc-293072, Santa Cruz Biotechnology), anti-NFκB (1:500) (sc-8008, Santa Cruz Biotechnology), anti-NALP3 (sc-134306, Santa Cruz, Biotechnology), anti-IL-1β (1:200) (NB600-633, Novus), and β-actin as loading control (1:750, Santa Cruz Biotechnology). After five washings with 0.1% Tween-20 in PBS, the membranes were incubated for 1 h at room temperature with peroxidase-conjugated secondary antibody anti-mouse (A90-116P Goat anti-mouse) and rabbit (A 120-101P Goat anti-rabbit) 1:5000 diluted in 1X PBS, 2.5% milk, and Tween-20 at 0.1% [23 (link)]. The levels of expression of the protein were detected using the enhanced chemiluminescence exposure process (ECL) (Amersham Pharmacia Biotech, Milan, Italy) with an image documenter Alliance 2.7 (Uvitec, Cambridge, UK). The detected signals were analyzed by ECL enhancement and assessed through UVIband-1D gel analysis (Uvitec). The data obtained were normalized with values assessed by densitometric analysis of the β-actin protein.

WB was performed according to our previous study method [17 (link), 35 (link)]. We used the following primary antibodies to perform the WB analyses: rabbit monoclonal anti-NF-κB p65 (D14E12) XP® antibody (1:1000, #8242, Cell Signaling Technology); rabbit polyclonal anti-Nrf2 (L593) antibody (1:500, BS1258, Bioworld); mouse monoclonal anti-Cryopyrin (NLRP3) (6F12) antibody (1:1000, sc-134306, Santa Cruz Biotechnology); rabbit polyclonal anti-PYCARD (ASC) antibody (1:500, A1170, Abclonal); goat polyclonal anti-caspase-1 p20 (M-19) antibody(1:1000, sc-1218, Santa Cruz Biotechnology); rabbit polyclonal anti-IL-1β (H-153) antibody (1: 1000, sc-7884, Santa Cruz Biotechnology); and rabbit polyclonal anti-IL-18 (H-173) antibody (1:1000, sc-7954, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Cell Signaling Technology) was used as an internal reference. Blot bands were quantified via densitometry with ImageJ software (National Institutes of Health, Baltimore, MD, USA), and protein levels were expressed as the ratio of values of the detected protein bands to that of GAPDH bands.