Bovine serum albumin (bsa)

MDCK cells were inoculated with original co-infected specimens. At 1 day after inoculation, the cells were fixed with 1 mL 3.7% formalin in PBS for 5 min, and then 1 mL cold methanol was added for 5 min. Cells were blocked by using 1 mL 1% bovine serum albumin (Serva, Heidelberg, Germany) in PBS-Tween 20 at 37°C for 1 hour. Rat and chicken antiserum against H5N1 and H9N2 viruses, respectively, were incubated with the fixed cells. Fluorescein isothiocyanate–conjugated goat anti–chicken IgG and goat anti–rat IgG diluted 1:2000 (KPL, Gaithersburg, MD, USA) were then added. Fluorescently labeled cells were examined by using fluorescence microscopy.

Transfected cells were harvested and lysed and the protein concentration was measured using a Bradford protein assay (Bio-Rad Laboratories, Feldkirchen, Germany). Bovine serum albumin (BSA) (SERVA Electrophoresis, Heidelberg, Germany) was used as standard. The cell extracts were separated through a 7.5% and 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Feldkirchen, Germany). After blocking with 5% BSA in tris-buffered saline (TBS) (0.1% Tween 20; 1% BSA) for 1 h, the membrane was incubated with the indicated primary antibodies (dilution 1:100) in TBS (0.1%, Tween 20; 1% BSA) overnight followed by their corresponding secondary antibodies. The protein expression was visualized by enhanced chemiluminescence (ECL) western blotting substrate (Bio-Rad Laboratories, Feldkirchen, Germany) in an ECL ChemoCam Imager (Intas, Göttingen, Germany). The intensity of the measured signals was quantified using ImageJ software and normalized to the loading control (α-tubulin).

Hela cells, obtained from ATCC (CCL-2), were grown in DMEM high glucose medium (Gibco) supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum (Gibco) and an additional 2 mM GlutaMAX (Gibco). For the interferon-gamma (IFNg) stimulation experiments, Hela cells were treated with 10 ng/mL recombinant IFNg (Cell Signaling), diluted in 0.5% (w/v) bovine serum albumin (Serva). For the preparation of the SILAC benchmark samples, Hela cells were grown in high glucose DMEM, with the previously listed supplements, dialyzed fetal bovine serum (Gibco) and heavy- (¹³C₆¹⁵N₄-Arg, ¹³C₆¹⁵N₂-Lys), intermediate (¹³C₆-Arg, D₄-Lys) or light isotope-containing Lysine, Arginine for 10 days. Hela cells were grown in 15-cm dishes for the preparation of newly synthesized proteome samples used for protocol optimization. For the SILAC benchmark sample preparation, the cells were grown in 10-cm dishes and for the preparation of the samples of IFNg-treated Hela cells, the cells were grown in 6-well plates. No cell line authentication and testing for Mycoplasma contaminations was carried out.

For antibody staining, cells were fixed with ice-cold methanol (Carl Roth) for 5 min at −20 °C, air dried for 15 min at room temperature, and rehydrated in PBS for 5 min. Detailed information on antibodies and controls is provided in Table S1. Blocking reagents and antibodies were diluted in PBS supplemented with 1.5% bovine serum albumin (w/v, Serva, Heidelberg, Germany). Cells on coverslips were incubated with primary and secondary antibodies for 45 min each. For the staining of cells on polyacrylamide gels, incubation times were prolonged to 24–72 h. Primary antibodies were incubated overnight at 4 °C and secondary antibodies were incubated in the dark at room temperature or at 4 °C. For nuclear staining, 1 µg/mL Hoechst 33342 (Sigma-Aldrich) was added to the staining solution of the secondary antibody. After each incubation step, cells were washed with PBS 3× for 10 min. Stained structures were briefly rinsed with H₂O and mounted with Mowiol 4-88 (Sigma-Aldrich). For the staining of F-actin, cells were fixed with 4% paraformaldehyde (Carl Roth) in PBS for 20 min at room temperature. Cells were washed 3× in PBS and permeabilized for 3 min with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. F-actin was labeled by overnight incubation with phalloidin that had been tagged with Alexa 488 (Thermo Fisher Scientific) and diluted 1:200 in PBS.

The inhibition study of hyaluronidase was set by a turbidimetric method described previously [13 (link),33 (link)]. The assay started by adding 20 µL of tested compound in monosodium phosphate buffer and then 40 µL of hyaluronidase solution (22.5 U/mL, Sigma Aldrich, Poznan, Poland) to the wells of 96-wells plates. The mixture was kept in the dark for 10 min at the temperature 37 °C. After incubation, 40 µL of hyaluronic acid solution (0.03%, Sigma Aldrich) in monosodium phosphate buffer was added to the wells. The mixture was kept in the dark for 45 min at the temperature of 37 °C. Finally, 300 µL of bovine serum albumin (0.1%, Serva) in sodium acetate buffer was added to the mixture and incubated for 10 min at room temperature. Changes in turbidity were measured by a microplate reader (BioTek, Winooski, VT, USA) at 600 nm. Heparin (WZF, Polfa, Warsaw, Poland) was a positive control. The assay was carried out in triplicate. The inhibitory activity of the tested compound was calculated from the equation [13 (link),33 (link)]: $\%\mathrm{inhibition}=100x\left(1-\left(\frac{A_{HA}-A_{AN}}{A_{HA}-A_{HYAL}}\right)\right)$ where A_HA—absorbance of solution without the enzyme (positive control), A_HYAL—absorbance of solution without the tested compound (negative control), A_AN—absorbance of solution with the tested compound.

Isolation of mononuclear blood cells was described previously in detail [28 (link), 29 (link)]. Briefly, recipients were anaesthetized and heparinized by i.v. injection of 200 U of heparin (Liquemin N 5000, Roche, Basel, Switzerland), and kidneys were intensively perfused with cold Ca²⁺- and Mg²⁺-free phosphate buffered saline (PAA, Pasching, Austria), supplemented with 2.7 mM EDTA and 0.1% bovine serum albumin (Serva, Heidelberg, Germany). To deplete erythrocytes and granulocytes, perfusates were purified by Percoll density centrifugation as described previously [28 (link)]. Mononuclear cells were counted and stored under liquid nitrogen until use.

The setup of the in vitro encrustation system is shown in Figure 1. Chemically defined synthetic urine was prepared according to Griffith et al. [8 (link)]. In brief, Solutions A and B were prepared with distinct amounts of different salt components. Solution A was supplemented with 0.3 U/mL urease (Carl Roth GmbH, Karlsruhe, Germany) to induce the crystallization process. Three hundred thirty three milligrams per liter of bovine serum albumin (Serva Electrophoresis GmbH, Heidelberg, Germany) were added to Solution B as a protein component. The temperature in the incubation system was set to 37 °C. The flow rate (Ismatec IPC pump system, Cole Parmer GmbH, Wertheim, Germany) was adjusted to 150 µL/min, with stop-go cycles of 50 s/10 s each to obtain measurable encrustations after 5 days in the encrustation system. The encrustations were analyzed quantitatively by measuring the weight of the samples before and after the experiment. The samples were allowed to dry for 3 d at 37 °C to ensure that all residual liquid was evaporated.