Bicinchoninic acid bca assay

Synthetic lipoxin A₄ (LXA4) was purchased from Cayman Chemical. Dulbecco's modified eagle's medium (DMEM/F‐12), RPMI‐1640 medium, penicillin–streptomycin (PS), and trypsin–EDTA solution were all purchased from Gibco®, Thermo Fisher Scientific. Fetal bovine serum (FBS), phosphate‐buffered saline (PBS) tablets, bovine serum albumin (BSA), alamarBlue™ reagent, Pierce™ IP lysis buffer, and bicinchoninic acid (BCA) assay were all purchased from Sigma‐Aldrich. Commercially available preparations of LPS from E. Coli were purchased from InvivoGen. TNFα and IL‐4 ELISA kits were purchased from R&D systems. Millicell® EZ 8‐well glass slides were purchased from Merk. All cell culture flasks and plates were purchased from Greiner Bio‐one.

Protein extracts from kidney and liver tissue were isolated using a radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholic acid, and 0.1% SDS. Proteins were boiled for 5 min, separated by 10–15% SDS-PAGE, and blotted onto polyvinylidene difluoride membranes. Proteins expression was detected using the following primary antibodies: PKLR, PFK-1, G6PC, PEPCK (1:1,000) (Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:10,000) (Sigma, USA). Protein bands were detected using an Immobilon Western Chemiluminescent HRP substrate kit (Millipore Corporation, Billerica, MA, USA). The image analysis program ImageJ (NIH; Bethesda, MD) was used for band intensity analysis. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Sigma-Aldrich).

Cells were lysed in RIPA buffer [150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 5.0 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, 0.5 mM dithiothreitol (DTT) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche) and PhosSTOP phosphatase inhibitor (Sigma-Aldrich)]. Extracts were sonicated and protein concentrations were determined with a bicinchoninic acid (BCA) assay (Sigma-Aldrich). Proteins were separated by SDS-PAGE [sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis] and transferred to nitrocellulose membranes by electroblotting. Primary antibodies for immunodetection were anti-Sox9 (Abcam, Cambridge, United Kingdom; ab3697) and anti-Tubulin (Sigma-Aldrich; T6074). Bound primary antibodies were detected using immunoglobulins conjugated with HRP (horseradish peroxidase; DakoCytomation, Glostrup, Denmark) and visualized by enhanced chemoluminescence (ECL). ECL signals were quantified using ImageJ 1.46f software (Figure 1B). Relative differences in Sox9 levels, corrected for background and Tubulin levels, were determined as compared to t = 0 conditions.

The mitochondrial fraction was isolated using the Cytochrome C Releasing Apoptosis Assay Kit (Abcam), according to the manufacturer's instructions. Briefly, cells were centrifuged at 600 g at 4°C, pellets were washed in ice-cold 1× PBS and centrifuged again. Supernatant was discarded and cells re-suspended in 1× Cytosol Extraction Buffer Mix (CEB), plus 1 mM DTT and 1% PIC. Samples were incubated on ice and then centrifuged at 10.000 g at 4°C. Supernatant was collected as cytosolic fraction. The remaining pellet was re-suspended in Mitochondrial Extraction Buffer Mix (MEB), plus 1 mM DTT and 1% PIC, and stored as mitochondrial fraction.
The protein content of each extracted fraction was then quantified by bicinchoninic acid (BCA) assay (Sigma-Aldrich).

The SS model was induced by immunization with SG autoantigen as previously described with some modifications [10 (link),17 (link),18 (link)]. Five C57BL/6 mice were sacrificed under an overdose of pentobarbital. The bilateral SGs of the mice were immediately removed under sterile conditions, dissected free from the surrounding fat and connective tissues and weighed. The bilateral SG was homogenized in 2 mL of sterile saline solution per 100 mg of SG and then centrifuged at 3000 g for 15 min at 4 °C. The supernatant was collected, and the protein concentration of the supernatant was determined using the bicinchoninic acid (BCA) assay (Sigma-Aldrich, Buchs, Switzerland), and then adjusted to 800 μg of protein per 1 mL of PBS, and emulsified in an equal volume of complete Freund’s adjuvant (CFA, Sigma–Aldrich, St. Louis, MO, USA) to a concentration of 400 μg of protein per 1 mL of solution. On day 0, each of the mice was injected subcutaneously with 0.1 mL of the emulsion. On day 14, the booster injection was carried out with the same dose of autoantigen emulsified in Freund’s incomplete adjuvant (IFA, Sigma–Aldrich, St. Louis, MO, USA). Control mice (naïve group) were immunized with 0.1 mL of PBS per mouse on days 0 and 14.

The EPO activity assay is a well-established and accurate method for measuring the number of eosinophils in biological samples, as described previously^{16 (link),17 (link)}. This method allows quantification of the reaction of peroxidases secreted by eosinophils through an optical absorbance measurement^{18 (link)}. Briefly, one lobe of the right lung was homogenized in 5% Hank’s Balanced Salt Solution (HBSS) and centrifuged at 960 × g for 10 min at 4 °C. After red blood cells lysis, we added 5% HBSS containing 0.5% hexadecyltrimethyl ammonium bromide to samples and performed three successive cycles of freezing and thawing. After centrifugation (1090 × g for 10 min at 4 °C), the samples were placed (75 μL/well) in a 96-well plate, and then, we added 150 μL of substrate (1.5 mM o-phenylenediamine and 6.6 mM hydrogen peroxide in 0.05 M Tris-HCl, pH 8.0) and incubated for 30 min at room temperature. Then, the reaction was stopped by the addition of 75 μL of 4 M sulfuric acid and the absorbance was read at 492 nm (Spectra Max M5, Molecular Devices, Sunny vale, CA, USA). The results are represented as optical density (OD) per ρg of protein in each sample. Protein was quantified by the Bicinchoninic acid (BCA) assay (Sigma-Aldrich Corp).