Trichloroacetic acid

Nessler reagent was purchased from Nacalai Tesque (Kyoto, Japan). Trichloroacetic acid and Folin–Ciocalteu’s phenol reagent were from Wako Pure Chemicals (Tokyo, Japan). The other reagents were chemically pure grades of commercial products.

The bile salt hydrolase activity of the recombinant LcBSH and GfBSH proteins was assayed, as previously described [16 (link),24 (link),25 (link),55 (link)]. Purified proteins were mixed with 0.24 mg/100 μL of conjugated bile salt solutions and incubated at 37 °C. In addition, each bile salt solution was mixed with a buffer solution (20 mM Tris, 150 mM NaCl, and 5% glycerol) instead of purified proteins and used as a negative control. The enzymatic reactions were terminated by the addition of 15% trichloroacetic acid (FUJIFILM Wako Pure Chemical Corporation). The denatured proteins were removed by centrifugation at 10,000× g for 15 min at 20 °C. The supernatant was then reacted with 300 mM borate buffer containing 1% SDS (pH 9.5) and 0.3% 2,4,6-trinitrobenzenesulfonic acid solution (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan). The reaction mixtures were statically incubated for 30 min at room temperature under dark conditions; finally, 0.6 mM HCl was added to stop the reaction. Absorbance at 416 nm was measured using a SPARK 10M multimode microplate reader (TECAN, Männedorf, Switzerland). The assays were performed in triplicate. The Student’s t-test was used to assess the presence of statistically significant differences using GraphPad Prism software (version 8.0; GraphPad Software, San Diego, CA, USA).

Glycogen contents in the liver and soleus muscle were measured using the Phenol-sulfuric acid method. First, 50 or 100 mg of liver or soleus muscle, respectively, were homogenized in 0.8 mL of 10% trichloroacetic acid (Wako Pure Chemical Industries Ltd.), followed by centrifugation at 1,900 × g for 10 min for deproteinization. The supernatant (0.4 mL) was mixed with 0.8% ethanol (Wako Pure Chemical Industries Ltd.), followed by centrifugation at 1,900 × g for 10 min to precipitate glycogen. Then, the supernatant was decanted and the precipitated glycogen was r-suspended in 0.5 mL distilled water. Phenol (0.5 mL of 5% stock; Wako Pure Chemical Industries Ltd.) and 2.5 mL concentrated sulfuric acid (Kanto Chemical Co., Inc., Tokyo, Japan) were added to the resuspended glycogen solution, and the mixture was incubated for 20 min at room temperature (20–22 °C). Then, the absorbance was measured at 490 nm using a U-5100 spectrophotometer (Hitachi High-Tech Science Co., Tokyo, Japan). A standard curve was generated using a 40 mg/dL glucose (Wako Pure Chemical Industries Ltd.) solution.

Cytoplasmic and nuclear lysates were prepared as previously described [24 (link)]. To prepare total cell lysates from granulocytes, the cells were precipitated in 10% trichloroacetic acid (Wako) for 30 min on ice. The TCA-precipitated fraction was treated with a lysis solution containing 9 M urea, 2% Triton X-100, and 1% dithiothreitol, and was disrupted by ultrasonication, followed by an addition of 2% lithium dodecyl sulfate and further ultrasonication. The proteins were separated on a 10% polyacrylamide gel, transferred onto Immobilon-P Transfer Membranes (Millipore, Billerica, MA, USA), and blotted with rabbit polyclonal anti-c-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit polyclonal anti-actin (Sigma), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz). Signals were detected by ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK).

Intracellular cGMP concentrations were measured in cells and rat lung tissues as described previously with some modifications.^{11 (link)} In the in vitro assay using SeV, SeV-infected cells were serum-starved for 6 hours or not starved on the day of the cGMP assay and were then incubated for 10 minutes with 1 mmol/l 3-isobutyl-1-methylxanthine (Wako, Tokyo, Japan) in order to inhibit the degradation of cGMP by PDE. Cells were subsequently incubated with 1 × 10⁻⁶ M CNP-22 (Bachem AG, Bubendorf, Switzerland) or vehicle (water) for another 10 minutes. The reaction was terminated with 100 µl of 0.1 mol/l HCl.
In the in vitro assay using sildenafil (5 µmol/l) or riociguat (100 µmol/l), on the day of cGMP assay (24 hours after drug stimulation), drug-treated cells were incubated for 20 minutes with 1 mmol/l 3-isobutyl-1-methylxanthine in order to inhibit the degradation of cGMP by PDE. The reaction was terminated with 100 µl of 0.1 mol/l HCl.
In the in vivo assay, fresh rat lungs were placed in 5% trichloroacetic acid (Wako), fractured by MicroSmash MS-100 (TOMY, Tokyo, Japan), and then centrifuged at 15,000 rpm for 10 minutes.
All cGMP concentrations were measured using the competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. cGMP concentrations in lung tissues were normalized by the protein concentrations of the samples.