Complete mini protease inhibitor cocktail

Five C57BL/6 mice per group were infected and sacrificed as described above. The bladders were obtained and weighed, sectioned five times, and sonicated in 500 μl of PBS with a pulse of 10 s and 70% amplitude (ultrasonic processor; Cole-Parmer; IL; US). Both kidneys were also obtained and weighed, and they were sectioned 10 times and sonicated in 500 μl of PBS with three pulses of 10 s and 70% amplitude. In both cases, they were supplemented with cOmplete™, Mini Protease Inhibitor Cocktail (Merck KGaA, Darmstadt, Germany). The organ lysates were centrifuged for 10 min at 4,000 rpm, and the supernatants were divided into aliquots in microtubes and stored at −70°C until use. The cytokines included in this study were IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and tumor necrosis factor (TNF). The cytokine release was quantified using a BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit (Becton, Dickinson Company, BD Biosciences, CA, USA) and a BD FACSCalibur™ flow cytometer. PBS was used as a control of cytokine basal release in this assay, and the wild-type strain was considered to show 100% cytokine release in a UTI. Data analysis was executed with FCAP Array software version 3.0 of BD Biosciences and FlowJo software version 7.6 (FlowJo, LLC, OR, USA).

Protein lysates were generated for each cell line by lysing cell pellets in RIPA buffer with 1% (v/v) phosphatase inhibitor (Phosphatase Inhibitor Cocktail 3; Merck, Gillingham, Dorset, UK) and 10% (v/v) protease inhibitor (cOmplete, Mini Protease Inhibitor Cocktail; Merck). Protein concentrations were measured using the Pierce BCA protein assay (ThermoFisher Scientific) according to the manufacturer's instructions. Western blots were run with 20 µg of protein per sample. Antibodies used are listed in Table 1 with cyclophilin B used as a loading control.³² (link) Original membrane images shown in Supplementary Figures S1, S2, and S3.

The concentration of cytosolic PAD4 in neutrophils was measured using the Human PADI4 ELISA kit (MyBioSource, San Diego, USA) according to the manufacturer’s instructions. Protein extraction was carried out using RIPA Buffer with cOmplete^™ Mini Protease Inhibitor Cocktail (Merck KGaA, Darmstadt, Germany). The protein levels were estimated from a generated standard curve. The optical densities were examined at a 450-nm wavelength using a microplate reader (Molecular Devices, Sunnyvale, USA).

See Supplementary Table 4 for a complete list of antibodies and dilutions used. Ba/F3 cells were lysed in Cell Extraction Buffer (Sample Diluent Concentrate 2, Bio-Techne), and patient-derived cells were lysed in radioimmunoprecipitation buffer; lysis buffers were supplemented with phosphatase (PhosSTOP) and protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail), both obtained from Merck. Total cellular proteins (10 µg for Ba/F3 cells, 20 µg for Ba/F3 xenografts or 25 µg for other cells) were subjected to SDS–PAGE. After electrophoresis, the separated proteins were transferred to PVDF membranes (Bio-Rad Laboratories), and then membranes were blocked in Blocking One-P (Nacalai Tesque), before incubation overnight with primary antibodies on a shaker in a cold room. The next day, membranes were washed and then soaked with HRP-linked anti-rabbit IgG (Cell Signaling Technology). The bands of the target proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) by the Amersham Imager 600 QC (Cytiva) or exposed to X-ray film and visualized using a Kodak X-ray developer.

See Supplementary Table 4 for a complete list of antibodies and dilutions used. Ba/F3 cells were lysed in Cell Extraction Buffer (Sample Diluent Concentrate 2, Bio-Techne), and patient-derived cells were lysed in radioimmunoprecipitation buffer; lysis buffers were supplemented with phosphatase (PhosSTOP) and protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail), both obtained from Merck. Total cellular proteins (10 µg for Ba/F3 cells, 20 µg for Ba/F3 xenografts or 25 µg for other cells) were subjected to SDS–PAGE. After electrophoresis, the separated proteins were transferred to PVDF membranes (Bio-Rad Laboratories), and then membranes were blocked in Blocking One-P (Nacalai Tesque), before incubation overnight with primary antibodies on a shaker in a cold room. The next day, membranes were washed and then soaked with HRP-linked anti-rabbit IgG (Cell Signaling Technology). The bands of the target proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) by the Amersham Imager 600 QC (Cytiva) or exposed to X-ray film and visualized using a Kodak X-ray developer.

Cells were lysed in a modified RIPA buffer (50 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 1 mM EGTA, 0.1% SDS, 50 mM NaF, 10 mM sodium β-glycerolphosphate, 5 mM sodium pyrophosphate, and 1 mM sodium orthovanadate and supplemented with Complete Mini Protease Inhibitor cocktail (Merck)), flash-frozen in liquid nitrogen and cleared via centrifugation. The samples were run for separation on 4–12% BisTris NuPAGE pre-cast gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes by Trans-Blot Turbo Transfer System (BioRad). The membranes were then blocked for 1 h in 5% milk or BSA in TBS-T (100 mM NaCl, 10 mM Tris, pH 7.6, 0.1% Tween 20) and cut prior to incubation with primary antibodies at appropriate dilution overnight at 4 °C. After washing with TBS-T the membranes were incubated in corresponding horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 1 h. Proteins on the membranes were detected via chemoluminescence induced by ECL detection reagent (GE Healthcare) or SuperSignal Western blotting reagent (ThermoFisher Scientific) using the ChemiDoc Imaging system (Biorad). The blots were quantified using Image Lab version 6.1 software (BioRad) and the data were analyzed using Prism GraphPad version 8. Full-length blots of blots used in figures are presented in Supplementary Fig. S7.