Af6423

Total protein was extracted from myocardial tissues by using RIPA lysis buffer (Santa Cruz Biotechnology, Texas, USA). After the protein concentration was determined, equal amounts (20 µg) of protein were mixed with 5 × loading buffer. Then, the mixture was separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). After that, the membranes were incubated with 5% nonfat milk for 1 h at room temperature. Subsequently, the membranes were incubated with antibody working solutions containing CPT-1 (1:2000, DF12004, Affinity), GLUT4 (1:2000, AF5386, Affinity), AMPK (1:2000, AF6423, Affinity), p-AMPK (1:2000, AF3423, Affinity), PGC-1α (1:2000, AF5395, Affinity), NRF-1 (1:2000, AF5298, Affinity), mtTFA (1:2000, AF0531, Affinity), ATP5D (1:2000, 14,893, ptgcn) at 4 °C overnight. The next day, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were incubated with ECL reagent (Chengdu ZEN Bioscience Co., Ltd., Chengdu, China) to visualize the protein bands. The relative expression of target proteins was normalized to β-actin. Immunoblot band intensities were quantified using NIH ImageJ software.

Briefly, paraffin sections (4 μm) were stained with primary antibodies, including anti-phospho-AMPKα (Thr172) (1:200, AF3423), anti-AMPKα (1:100, AF6423), anti-SIRT1 (1:100, DF6033) and anti-PGC-1α (1:100, AF5395), at 4°C overnight, which were purchased from Affinity. Positive staining was visualized using a DAB color development kit (G1211, Servicebio). Finally, the dyed sections were photographed with an optical microscope, and the positive staining area was calculated by ImageJ software.

LMH cell line (ATCC^® CRL-2117™) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, San Diego, CA, United States), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO₂. The virulent FAdV-4 strain SDDM-4/15 used in the study was isolated from clinical samples of dead chickens and characterized by our research group (Niu et al., 2016 (link)). A γ-secretase inhibitor (DAPT) were purchased from Sigma (St. Louis, MO, United States), and reconstituted in dimethyl sulfoxide and stored in −80°C. The primary antibodies used in the study were specific for GAPDH (Goodhere, AB-P-R001), Hes1 (Affinity Biosciences, DF7569), AMPK (Affinity Biosciences, AF6423).

For intracellular AMPK activity analysis, HepG2 cells were seeded in six-well plates and grown to 50% confluence with high-glucose DMEM followed by a 6 h free-serum quench. Then, cells were treated with VTE (0, 60, 125, 250, and 500 μg ml⁻¹) for 24 h. Then cells were collected for Western blotting.
The whole-protein extractions from HepG2 cell and livers of mice were separated using 10% SDS-PAGE and then transferred to nitrocellulose membranes. The expression of protein was subsequently probed with primary antibodies of AMPK (Affinity Biosciences, AF6423), phosphorylation of AMPK (p-AMPK, Affinity Biosciences, AF3423), Cleaved Caspase-3 (cle-CASP3, Affinity Biosciences, AF7022), CHOP (Proteintech Group, 15204-1-AP) and β-ACTIN (Proteintech Group, 60008-1-Ig) with 1:1,000 dilution in 5% bovine albumin at 4°C overnight followed by HRP-labelled secondary antibodies (1:10,000, Yeasen, Shanghai, China) for 1 h at room temperature. The protein bands were detected and analysed using the Tanon-5200 Multi-image detection system and ImageJ software respectively. The levels of relative protein were normalized to β-ACTIN.

The extracted protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. After being blocked, the membranes were incubated with rabbit polyclonal anti-AMPK (1:1000, AF6423, Affinity Biosciences), rabbit polyclonal anti-p-AMPK (1:1000, AF3423, Affinity Biosciences), rabbit polyclonal anti-RIPK1 (1:1000, AF7877, Affinity Biosciences), rabbit polyclonal anti-RIPK3 (1:1000, AF7942, Affinity Biosciences), rabbit polyclonal anti-MLKL (1:1000, DF7412, Affinity Biosciences), rabbit polyclonal anti-HMGB-1 (1:1000, AF7020, Affinity Biosciences) and rabbit polyclonal anti-β-actin (1:1000, AF7018, Affinity Biosciences) antibodies at 4 ℃ overnight. Then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies (1:1000; SA00001-2, Proteintech) for 2 h. Protein band intensity was quantified with ImageJ software.

We selected six time points (0, 15, 30, 60, 120, and 240 min) to determine the peak time of activation of the AMPK signaling pathway following the stimulation of cultured cells with 10 ng/mL IL-1β using western blotting. Furthermore, we cotreated the cultured cells with 2 μM AdipoRon and 10 ng/mL IL-1β and evaluated the effects of AdipoRon on the AMPK/NF-κB signaling pathway at the peak time point. In addition, western blotting was performed six times using six patient samples to evaluate AMPK phosphorylation. Primary antibodies against AMPK (AF6423; Affinity Biosciences, Cincinnati, OH, USA), anti-phospho-AMPK (Thr 172) (AF3423; Affinity Biosciences), anti-p65 (aAF5006; Affinity Biosciences), and anti-phospho-p65 (AF2006; Affinity Biosciences) were used. Representative immunoblots of six similar results are presented (n = 6) (Figure 9a).

Drugs were added to the BV2 microglia as previously described. Twelve hours after administration, cells were collected and cleaved; protein concentration was quantified; protein lysate (25 μL) was electrophoresed and transferred onto PVDF membrane (pore size: 0.45 μm). After the transmembrane was completed, the PVDF membrane was sealed with 5% skim milk for 2 h and incubated with the specified primary antibody—against P-AMPK (T55608S, 1:1000, Abmart, Shanghai, China), mTOR(T55306S, 1:5000, Abmart), P-mTOR (T56571S, 1:5000, Abmart), LC3-II/LCI (T55992F, 1:500, Abmart), IL-1β (TA5103, 1:500, Abmart), P62 (T55546S, 1:5000, Abmart), NLRP3 (15101s, 1:1000, Cell Signaling Technology (CST), Danvers, MA, USA), GAPDH (T0004, 1:1000, Affinity, Cincinnati, OH, USA), caspase-1 p20 (AF4005, 1:1000, Affinity) or AMPK (AF6423, 1:1000, Affinity)—at 4 °C overnight. PVDF membrane and HRP-labeled secondary antibody were cultured at room temperature for 1 h and rinsed 3 times. Visualization of the protein bands was achieved with an enhanced chemiluminescence (ECL) kit (P0018FS, Vazyme, Nanjing, China) and imaging system (3500R, Tanon, Shanghai, China), whose grayscale values were calculated using ImageJ version 5.0.