Af3423

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.

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 animals were anesthetized at 24 h after surgery, and the brains were fixed using left ventricle perfusion with normal saline, then incubation in 4% paraformaldehyde. The sections were deparaffinized by xylene, rehydrated with different concentrations of ethanol solutions, and treated with antigen regeneration using 10 mM sodium citrate buffer (pH 6.0). After blocked with normal goat serum at 37 °C for 30 min, the sections were then incubated with primary antibodies as follows by overnight incubation at 4 °C: Nampt (1:50; 11776-1-AP, Proteintech, China), p-AMPK (1:100; AF3423, Affinity, USA), and p-mTOR (1:100; AF3308, Affinity, USA) antibodies. Then, the sections were incubated with corresponding biotinylated secondary antibody at 37 °C for 30 min. Positive staining was determined after treatment with 3-3′diaminobenzidine (D8001, Sigma-Aldrich, USA). Finally, the sections were observed using a DM600B automatic microscope (Leica Microsystems, Heidelberg, Germany) and photographed, and then quantified by ImageJ software (National Institutes of Health).

As mentioned above [14 (link)], Western blot analysis was used to quantify protein expression levels in colon tissues. The primary antibodies and their dilution concentrations were as follows: Phospho-AMPK (1:1000, AF3423, Affinity Bioscience), AMPK (1:5000, 10929-2-AP, Proteintech), SIRT1 (1:1000, A17307, ABclonal), NLRP3 (1:2000, A12694, ABclonal), caspase-1 (1:1000, A16792, ABclonal), iNOS (1:1000, A3200, ABclonal), Arg1 (1:1000, A1847, ABclonal), NOX2 (1:1000, A19701, ABclonal), and HADHB (1:1000, A5716, ABclonal). β-actin (1:1000, GB12001, Servicebio, China) was used as the internal reference. HRP Goat Anti-Rabbit IgG (1:10 000, AS014, ABclonal) was used as the secondary antibody. We used ImageJ Software to analyze the protein band results.

Paraffin-embedded TB patient lung and LN tissues were provided and sliced into 5 μm–thick sections by Guangzhou Chest Hospital with the written consent of all participants. Levels of ZNFX1 in vivo were detected with immunohistochemistry (IHC) staining according to the previously described protocol (57 (link)), with the antibody against ZNFX1 (1:50; catalog HPA046629; Merck Millipore) and HRP-conjugated goat anti-rabbit IgG (H+L) (1:200; catalog 31460; Thermo Fisher Scientific). Paraffin-embedded sections of murine lungs and spleens were stained with anti-F4/80 antibody (1:100; catalog MA5-16630; Thermo Fisher Scientific) together with anti-LC3 antibody (1:100; catalog 14600-1-AP; Proteintech) or anti–p-AMPK antibody (1:100; catalog AF3423; Affinity) at 4°C overnight, followed by staining with goat anti-mouse IgG 488 (catalog A28175) for F4/80 and goat anti-rabbit IgG 647 (catalog A27040) (Thermo Fisher Scientific) for LC3 and p-AMPK at room temperature for 1 hour. Samples were then scanned with CaseViewer2.4 (3DHISTECH). The levels of ZNFX1 in the TB tissues and the levels of LC3 puncta or p-AMPK in macrophages in murine lung and spleen tissues were determined using ImageJ 1.53 by a reader following a protocol blinded to the knockout versus WT experimental condition.

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.