Nlrp3 rabbit mab

Proteins were separated with SDS–PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. Blots were incubated with the following primary antibodies: A20/TNFAIP3 rabbit mAb (Cell Signaling Technology, 5630), phospho-IKKα (Ser176)/IKKβ (Ser177) rabbit mAb (Cell Signaling Technology, 2078), phospho-NF-κB p65 (Ser536) rabbit mAb (Cell Signaling Technology, 3033), IL-1β rabbit mAb (Cell Signaling Technology, 12703), NLRP3 rabbit mAb (Cell Signaling Technology, 15101), caspase-1 rabbit mAb (Abcam, ab207802), NF-kB p65 rabbit mAb (Abcam, ab32536), IKKβ rabbit mAb (Abcam, ab32135), and GAPDH mouse mAb (ZSGB-BIO, TA-08). Immunoblots were probed with goat anti-rabbit IgG (H&L)-HRP conjugated antibody (Easybio, BE0101) or goat anti-mouse IgG (H&L)-HRP conjugated antibody (Easybio, BE0102), developed using Chemiluminescent Western Blotting Substrate (Millipore, WBKLS).

The following antibodies were used for western blotting: NLRP3 rabbit mAb (#15101), ASC rabbit mAb (#67824), IL-1β mouse mAb (#12242), phospho-NF-κB p65 rabbit pAb (Ser536; #3033), phospho-IκBα mouse mAb (Ser32/36; #9246), total-IκBα rabbit mAb (#9242), phospho-mTOR rabbit mAb (Ser2448; #2971), total-mTOR rabbit mAb, phospho-p70 S6 Kinase rabbit mAb (Ser371; #9208), raptor rabbit mAb 46, DEPTOR rabbit mAb (#11816), PRAS40 rabbit mAb, anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Caspase-1 p10 rabbit pAb (sc-514), total-NF-κB p65 rabbit pAb (sc-7151), total-p70 S6 Kinase mouse mAb (sc-8418), and β-actin mouse mAb (sc-47778). All antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Protein lysates were extracted from brain tissues or culture cells by RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Sigma, USA). Protein concentration was measured by BCA kit (Beyotime, China). A total of 20 μg protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Biorad, USA). Then, the membranes were blocked by 5% fat-free milk for 1 h at room temperature, incubated with first antibodies at 4 °C overnight and corresponding second antibodies for 1 h at room temperature. Then antibodies used in our study were: iNOS Rabbit mAb (CST#13120, 1: 1000), Arginase-1 Rabbit mAb (CST#93668, 1: 1000), GAPDH Rabbit mAb (CST#2118, 1: 1000), LC3A/B Rabbit mAb (CST#12741, 1: 1000), p62 Antibody (CST#5114, 1: 1000), Phospho-Akt (Ser473) Antibody (CST#9271, 1: 1000), Akt Antibody (CST#9272, 1: 1000), Phospho-mTOR (Ser2448) Antibody (CST#2971, 1: 1000), mTOR Rabbit mAb (CST#2983, 1: 1000), pro-IL-1β Rabbit mAb (CST#31202, 1: 1000), Cleaved-IL-1β Rabbit mAb (CST#63124, 1: 1000), pro-Caspase-3 Antibody (CST#9662, 1: 1000), Cleaved Caspase-3 Antibody (CST#9661, 1: 1000), Phospho-GSK-3α/β (Ser21/9) Antibody (CST#9331, 1: 1000), GSK-3α/β Rabbit mAb (CST#5676, 1: 1000) and NLRP3 Rabbit mAb (CST#15101, 1: 1000) were all obtained from Cell Signaling Technology (USA).