Western blotting
Protein samples were electrophoresed on polyacrylamide gels and
transferred onto
PVDF (Merck, Germany) membranes. The membranes were blocked with 5%
skim milk in PBS and incubated with anti-ACSL4 (1:1000; Boster, Wuhan,
China), anti-GPX4 (1:1000; Abmart, Shanghai, China), anti-xCT (1:1000;
Boster, Wuhan, China), anti-IL-6 (1:1000; Abclonal, A1570, Shanghai,
China), anti-IL-17 (1:200; Abclonal, A10587, Shanghai, China), anti-10
(1:1000; Abclonal, A2171, Shanghai, China), anti-NLRP3 (1:1000;
Abclonal, A12694, Shanghai, China), anti-ASC (1:1000; Abclonal, A16672,
Shanghai, China), anti-Caspase 1 (1:300; Abclonal, A0964, Shanghai,
China), anti-ASC (1:1000; Abclonal, A16672, anti-IL-1β (1:1000;
Proteintech, Wuhan, China), anti-F4/80 (1:300; Cell Signaling
Technology, Q61549, USA), anti-CD86 (1:1000; Abclonal, A16805, Shanghai,
China), anti-iNOS (1:1000; Abclonal, A3774, Shanghai, China), anti-CD206
(1:1000; Cell Signaling Technology, Q61830, USA), anti-NF‑κB (1:1000;
Abclonal, A2547, Shanghai, China),
anti-IKB\(\alpha\ \left(1:1000;\ Abclonal,\ A1187,\ Shanghai,\ China\right),\ \)anti-p-IKBα
(1:1000, Abclonal, AP0707, Shanghai, China), anti-p-NF‑κB (1:1000;
Abclonal, AP0944, Shanghai, China), and anti-Cystatin C (1:1000; A8933,
Shanghai, China) at 4 °C overnight. After being washed with PBS-T (PBS
with 0.1% Tween-20), the membranes were incubated with secondary
antibodies at room temperature for 1 hour and washed three times with
PBST for 5 min each. Specific bands were visualized by a Tanon-5200
Multi Chemiluminescent System (Tanon, Shanghai, China) and
hypersensitive ECL luminescent liquid. The grayscale values were
measured and quantified using ImageJ software.