Anti asc

The levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, TRAF6, NLRP3, ASC, and caspase-1 were quantified by Western blotting. First, proteins were extracted by cutting and grinding tissue samples in RIPA lysis buffer. The lysates were then centrifuged, and the supernatants were collected. Protein concentrations were determined using a BCA protein quantitation kit (GeneCopoeia). Proteins were then separated by SDS–PAGE and subsequently transferred onto methanol-pretreated PVDF membranes (Millipore Corporation, USA). The membranes were then blocked with 5% skimmed milk powder at room temperature for 1 h and incubated with the following antibodies: anti-TLR4 rabbit pAb (Zen, 1 : 1,000); anti-TRAF6 rabbit pAb (Zen, 1 : 1,000); anti-TAK1 rabbit pAb (Zen, 1 : 1,000); anti-NF-κB-p65 mAb (Zen, 1 : 1,000); anti-MYD88 rabbit pAb (Zen, 1 : 800); anti-IRAK1 (PTG, 1 : 1,000); anti-ASC (Bioss, 1 : 1,000); anti-NLRP3 (Bioss, 1 : 1,500); and anti-caspase-1 (Bioss, 1 : 800). The membranes were subsequently incubated with the appropriate secondary antibodies for 1 h (HRP-conjugated goat anti-rabbit IgG (Ubio, 1 : 5,000) and HRP-conjugated goat anti-mouse IgG (Ubio, 1 : 5,000)). GAPDH and β-tubulin (Ubio) were used as reference proteins.

L-NAT was obtained from Sigma Aldrich (St. Louis, MO., USA). Rat ELISA kit was purchased from NeoBioscience (Shanghai, China). The cell counting kit-8 (CCK-8) came from 7sea-Biotech (Shanghai, China). Anti-ASC, NLRP3, IL-1β, TLR4 and NF-κB were purchased from Bioss Antibodies (Beijing, China). Anti-Caspase 1 antibody came from Santa Cruz Biotechnology (Shanghai, China). TRIzol reagent was obtained from Life Technologies (USA). RTPA lysis was purchased from SolarBio Life Sciences (Beijing, China).

Samples were subjected to 12% SDS-PAGE and were then transferred to nitrocellulose filter membranes. Membranes were blocked in 5% skim milk at 25 °C for 1.5 hours and were then incubated with the primary antibodies. Anti-Caspase-1 (1 µg/mL; BOSTER), anti-NLRP3 (1 µg/mL; BOSTER), anti-IL-1β (2 µg/mL; BIOSS, Shanghai, China), anti-IL-18 (0.76 µg/mL; Proteintech), anti-ASC (2 µg/mL; BIOSS, Shanghai, China), anti-PDCD6 (programmed cell death protein 6; 0.734 µg/mL; Proteintech), anti-Cleaved Caspase-1(1 µg/mL;Cell Signaling Technology), and anti-Actin (0.22 µg/mL; Proteintech) antibodies were used in this study. After 4 washes with PBS, membranes were incubated and reacted with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were developed with enhanced chemiluminescence reagents (Amersham, United Kingdom).