Sc1218

Hippocampus tissues were lysed using RIPA protein lysis buffer and total protein was collected by centrifugation. Protein concentration was determined using BCA method according to the manufacturer’s instructions. Protein samples (2 μg/μl) were subjected to SDS/PAGE and then transferred on to PVDF membranes. After transfer, membranes were incubated with the primary antibodies against NLRP3 (1:2000, ab51952, Abcam, U.K.), caspase-1 (1:1000, sc1218, Santa Cruz Biotechnology, U.S.A.), and β-actin (1:10000, 3700, Cell Signaling Technology, U.S.A.) at 4°C overnight and then incubated with secondary horseradish peroxidase-conjugated IgG antibodies (1:5000) at 37°C for 2 h. The protein bands were developed using the Beyo electrochemiluminescence Plus reagent and the images were captured in the Gel Imaging System (WD-9413B, Liuyi Factory, China).

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.