Anti txnip

The levels of inflammasome protein expression were assessed using western blot analysis. Five tumor tissues per group were analyzed in triplicate. Protein concentrations were measured using the Bradford assay (Thermo Fisher Scientific, Carlsbad, CA, USA), and equal amounts of protein from each sample were subjected to western blot analysis with anti-TXNIP (Santa Cruz Biotechnology, Dallas, TX, USA), anti-caspase-1, GAPDH (Cell Signaling Technology, Beverly, MA, USA), anti-NLRP3, anti-CTP1A, anti-ASC, and anti-IL-1β (Abcam, Cambridge, UK). Target proteins were normalized to the reference housekeeping gene GAPDH.

Western blot was performed as previously described.²³ Proteins were loaded into SDS‐PAGE gel, and electrophoresis was performed. Then, the proteins were transferred to a nitrocellulose membrane, which was blocked with 5% non‐fat blocking grade milk (Bio‐Rad, Hercules) for 1 hour at room temperature. The membranes were incubated with the primary antibodies overnight at 4°C. The following primary antibodies were used: anti‐TXNIP (1:500; Santa Cruz Biotechnology), anti‐NLRP3 (1:1000; Novus Biologicals, Centennial), anti‐cleaved caspase‐1 (1:1000, Novus Biologicals), anti‐interleukin‐1β (1:1000; Santa Cruz Biotechnology), anti‐TNF‐α (1:500; Santa Cruz Biotechnology) and anti‐interleukin‐6 (1:1000; Santa Cruz Biotechnology). Anti‐β‐actin (1:3000; Santa Cruz Biotechnology) was used as loading control. The membranes were then incubated with appropriate secondary antibodies for 2 hours at room temperature. Immunoreactive bands were visualized with an ECL Plus kit (Amersham Biosciences) followed by exposure to X‐ray films and analysed using ImageJ software (NIH, Bethesda). For PC12 cells, expression of TXNIP and secreted pro‐inflammatory cytokines were measured from 4 separate experiments and normalized to expression of β‐actin in the cell lysate.²⁴

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).