Kz 2

After perfusion with cold saline (0.9%), proteins from renal tissue were acquired using a tissue homogenizer (KZ-II; Servicebio). A total of 20 μg of protein was used for WB analysis, and 10% SDS-PAGE gels were used for WB. Polyvinylidene fluoride membranes (0.45 µm; IPVH00010; Millipore, Billerica, MA) were used for protein transfer. Primary antibodies used for WB were purchased from Cell Signaling Technology (CST). Antibodies, such as SAPK/JNK (9252; RRID: AB_2250373; CST), Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) (4668; RRID: AB_823588; CST), p38 MAPK (D13E1) (8690; RRID: AB_10999090; CST), Phospho-p38 MAPK (Thr180/Tyr182) (4511; RRID: AB_2139682; CST), p44/42 MAPK (Erk1/2) (4695; RRID: AB_390779; CST), and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370; RRID: AB_2315112; CST) were used to detect MAPK signaling pathways. GAPDH (D16H11) XP^® Rabbit mAb (HRP Conjugate) antibody was used for GAPDH detection (8884; RRID: AB_11129865; CST).

Cells were lysed using RIPA lysis buffer (# P0013K, Beyotime) supplemented with protease inhibitors and skin tissue was ground with a tissue lyser (#KZ-II, Servicebio). The protein content was measured with a BCA kit (#P0010, Beyotime). The lysate (10-20 ug protein) was separated by SDS- PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% skim milk. The membranes were probed with specific primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated in HRP-conjugated secondary antibody for 2 h at room temperature and visualized with an ECL reagent (# BL520A, Biosharp). Protein content quantification was evaluated with Image J software (NIH, USA).

MLs were obtained by artificially digesting T. spiralis-infected mouse muscles at 42 days post-infection (dpi). The IILs and AWs were recovered from the small intestine of infected mice at 6 h post-infection (hpi) and 2, 3 and 6 dpi, respectively [30 (link), 31 (link)]. Six-day-old female adults were cultured in RPMI-1640 with 10% foetal bovine serum (FBS; Gibco, New Zealand) at 37 °C in 5% CO₂ for 24 h, and the NBLs were harvested [32 (link)]. Soluble worm somatic proteins of various stage worms (MLs, IILs, AWs and NBLs), excretory/secretory antigens (ESA) from MLs, IILs and 6 d AWs were prepared as described previously [33 (link)]. Briefly, the diverse stage worms were first homogenized with a tissue grinder (KZ-II Servicebio), and worm tissue fragments were further homogenized using ultrasonication. The supernatant carrying worm soluble proteins was obtained after centrifugation at 15 000 × g for 1 h at 4 °C. Moreover, to prepare the ESA, the worms were washed using sterile saline and cultured in RPMI-1640 medium (5000 worms/mL) at 37 °C and 5% CO₂ for 18 h. The culture medium containing ESA was filtered through a 0.22 μm membrane and concentrated using an ultrafiltration tube. The concentration of soluble proteins and ESA was ascertained by a Coomassie brilliant blue G-250 method [17 (link)].

As previously described^{33 (link)}, the protein was extracted by grinding machine (KZ-II, Servicebio, Wuhan, CN) and radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor (Beyotime, Shanghai, China). The protein concentration was measured by the BCA Protein Assay kit (Beyotime).
After SDS-PAGE, the proteins were transferred to PVDF membranes (0.45 μm pores, Millipore, MA, USA). After blocking, the membranes were incubated with primary antibodies against p-IRS (1:500, Proteintech, USA), IRS (1:1000, Proteintech), p-AKT (1:2000, Proteintech), AKT (1:2000, proteintech), MIF (1:1000, Abcam, Cambridge, UK), p-AMPK (1:1000, Abcam), AMPK (1:1000, Abcam), SIRT1 (1:500, Proteintech), P62 (1:1000, Proteintech), p-ULK1 (1 µg/ml, Abcam), ULK1 (1 µg/ml, Abcam), LC3 (1:1000, Proteintech), BAD (1:1000, Abcam), BAX (1:1000, Abcam), Bcl-2 (1:1000, proteintech, USA), β-actin (1:5000, Proteintech) respectively. Following incubation of HRP-labeled secondary antibody (1:5000, Proteintech), the band pictures were collected and analyzed by the gel documentation system (Bio-Rad, CA, USA).

Seven-week-old female mice CD-1 were purchased from Hunan Slake Jingda Experimental Animal, Co., Ltd. (Hunan, China). They weighed approximately 25 ± 3 g at the time of the experiments.
The high bacterial load abscess infection model was performed as defined earlier with slight adaptations (Pletzer et al., 2018 (link)). Before the injection, bacterial cells were rinsed resuspended in 1 × PBS. An injection of bacterial suspension was given to the dorsum to achieve the concentrations to generate reproducible abscesses and bacterial counts: S. aureus, 1 × 10⁸ CFU/mice; and E. faecalis, 1 × 10⁹ CFU/mice. Antimicrobial administration was given directly into the subcutaneous space of the infected area at 1 h post-infection. The development of the infection was observed every day. Abscesses were determined on day 2 using a caliper. Skin abscesses were removed (comprising all accrued pus) and regimented in sterile PBS by an automatic tissue homogenizer (Servicebio KZ-II, Wuhan, China). Bacterial counts were quantified by serial dilution. For histopathological analyses, hematoxylin and eosin (H&E) staining was performed.

After LPS challenge for 24 h, the BALF was collected for the measurement of TNF-α and IL-1β. And the right upper lung was immediately frozen and homogenized in ice-cold PBS by a tissue grinder (Servicebio KZ-II, Wuhan, China) at 4°C for 10 min to obtain 10% homogenate. After centrifuged at 2,000g for 10 min at 4°C, the concentration of TNF-α and IL-1β in supernatant were measured by ELISA kits following the manufacturer’s procedures (Biolegend, San Diego, USA), normalized to total protein as assessed by a BCA kit (Thermo, MA, USA) as previously described (Maia et al., 2019 (link)).

The blood of the heart tissue was washed with PBS, and tissue blocks of appropriate size were cut out with scissors and put into EP tubes. A 4 mm large steel balls and 3 mm small steel balls were added to each tube, and an appropriate amount of tissue lysate was added for cracking. Samples were then homogenized in a high-speed tissue grinder (Servicebio KZ-II, China), and the supernatant was obtained by centrifugation as tissue protein. SDS-polyacrylamide gels with various concentrations of sodium dodecyl sulfate (SDS) were electrophoretically resolved, and PVDF membranes were electro-transferred. Different molecular weights of proteins were electrophoretically resolved with sodium dodecyl sulfate (SDS)-polyacrylamide gels, and polyvinylidene difluoride (PVDF) membranes were electro-transferred. Membranes were sealed with 5% skimmed milk for 1 h and then washed three times with PBST, each time for five minutes. Then, membranes were incubated with primary antibody overnight at 37 °C and then secondary antibody at room temperature for 1 h. Finally, samples were detected by the ECL color method.
For protein interaction (co-IP) experiments, the antibody and dynabeads were first mixed for 16–22 h. Then, the tissue lysate was added for 1 h. PBST was rinsed three times; then, the dynabeads were removed, after which western blotting was performed.