Immunoblotting Analysis of Liver Proteins

Protein samples were extracted from liver tissues and cultured cells as previously reported [28 (link)]. Equal amounts of protein (30–40 μg) were separated by 8%–15% SDS-PAGE and then transferred onto PVDF membranes. For immunoblot analysis of IL-1β levels in the cell culture medium, supernatants were collected for protein extraction as previously reported [35 (link)]. The following primary antibodies were used: anti-IL-1β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr¹⁷² (number 2535), anti-phospho-GSK3β Ser⁹ (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser⁷⁹ (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser⁴⁷³ (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA). Goat anti-rabbit (number A21020) and goat anti-mouse (number A21010) secondary antibodies were purchased from Abbkine (Redlands, CA, USA).