Bradford assay reagent

We pelleted cells from each condition at 5,000 g for 10 min. After the supernatant was decanted, the pellets were resuspended in 60 μl of B‐PER (Thermo Scientific) containing complete Mini ethylenediaminetetraacetic‐acid‐free protease inhibitor cocktail (Roche), and cells were lysed for 30 min at room temperature. After pelleting the cell debris by centrifugation at 15,000 g for 5 min, we transferred 40 μl of clear lysate to a new tube. We determined protein concentrations using a Bradford assay reagent (Thermo Scientific) and denatured proteins by adding 10% sodium dodecyl sulfate [SDS] (Sigma‐Aldrich) to a final concentration of 2%. Next, 60 μg of total protein from each cell lysate were loaded and separated by 12% SDS‐polyacrylamide gel electrophoresis. The proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon, 0.45 μm, Millipore) and probed via immunoblotting. We used the following antibodies for immunoblotting: rabbit anti‐TcpA (Taylor et al, 2004 (link); 1:2,500), mouse anti‐RnaP (Biolegend; 1:2,500), anti‐rabbit horseradish peroxidase‐conjugated (Promega, 1:2,500), and anti‐mouse horseradish peroxidase‐conjugated (Invitrogen; 1:2,500). The immunoblots were developed with the SuperSignal West Pico chemiluminescent kit (Pierce).

Male, 5-week-old Sprague-Dawley (SD) rats (Orientbio Inc.) were individually infected with 50 metacercariae of C. sinensis. At 8 weeks post-infection, the rats were euthanized under deep anesthesia with ethyl ether, before adult worms were isolated from the bile ducts, washed five times with phosphate-buffered saline (PBS) containing 100 U/ml penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA), then incubated for 24 h at 37°C with 5% CO₂. After incubation, the medium was centrifuged for 10 min at 1,000 × g to remove the worms and cellular debris. The supernatant was then centrifuged for a further 10 min at 18,000 × g before being filtered with a syringe-driven 0.45 μm filter. The concentration of protein was measured using Bradford assay reagent (Thermo Fisher Scientific, Waltham, MA, USA).

For experiment in cultured cells, cellular fractionation into cytosol and intact mitochondria was done essentially as previously described^{91 (link),92 (link)}. Briefly, mitochondria from BMDM pellets (~10⁷ cells) were isolated from the cytosolic fractions after cell permeabilization with a buffer containing 0.1% digitonin in 210 mM mannitol, 20 mM sucrose and 4 mM HEPES. The pellets after centrifugation at 700 × g for 5 min contained mitochondria.
Further differential centrifugation at 100,000 × g of these mitochondrial-enriched lysates was used to purify the mitochondrial matrix fraction. Swelling of the mitochondrial outer membrane was performed by hypotonic shock, followed by sonication (four repeats of 5 s- bursts at high settings with 30 s pauses), supplementation with 150 mM NaCl and 5% glycerol and centrifugation at 20,000 × g 30 min. The supernatant was saved as soluble mitochondrial matrix fraction.
For isolation of mitochondria from whole liver, homogenization was performed as previously described^{91 (link),92 (link)} with glass–Teflon potter for 4–5 strokes. Total protein (mg/mL) was determined using the Bradford Assay reagent (Thermo). Mitochondria were resuspended in MAS IX buffer and incubated 1 h at 25 °C with 500 μM Angeli’s salt or DEA/NO in gentle agitation. Then mitochondria were pelleted by centrifugation at 7000 × g, 10 min, 4 °C and used for subsequent assays.