Inhibitors of proteases

The cells were plated onto ∅ 30 mm tissue culture dish to reach density of approximately 70% on the following day. Transfection was performed with 4 μg of plasmid DNA (pcDNA 3.1.-NP and pcDNA3.1 empty vector) diluted in 400 μl of serum free medium and 6 μl of Turbofect transfection reagent (Thermo Scientific, Vilnius, Lithuania) and incubated for 20 min at room temperature. The reaction mixture was added drop-wise to the cells and the cells were incubated for 24 h at 37°C. The medium (3 ml DMEM with 10% FCS) was changed after 24 h and the following day, the cells were used for protein extraction. Protein extraction was done with 50 μl of ice-cold RIPA buffer (Sigma-Aldrich) supplemented with inhibitors of proteases (Roche Diagnostics GmbH, Mannheim, Germany) per culture dish and incubated on ice for 15 min. The cells were scraped into the eppendorf tube and centrifuged at 14,500 rpm for 15 min. Supernatant was transferred into new eppendorf tube and used for western blot analysis.

L-929 cells incubated for 48 h in normoxia or hypoxia were solubilized in ice-cold RIPA buffer (1% Triton X-100; 1% deoxycholate, 150 mM NaCl, pH 7.2) containing inhibitors of proteases (Roche Applied Science, Mannheim, Germany) for 30 min on ice. Cell lysates were collected, cleared by centrifugation at 10,000× g for 10 min at 4 °C. Protein concentration was determined using the BCA protein assay reagent (Pierce, Rockford, IL, USA). Total protein extracts were separated in 10% SDS-PAGE and transferred onto PVDF membrane (Immobilon^TM-P, Milliopore, Billerica, MA, USA). For mCA IX detection, the membrane was incubated with mouse monoclonal AM4-3 antibody (10 μg/mL) diluted in blocking buffer for 2 h. Secondary anti-mouse peroxidase-conjugated antibody (Sigma–Aldrich, St. Louis, MI, USA) was diluted 1:5000 in blocking buffer. For CD63 analysis, mouse monoclonal antibody (1 μg/mL, ThermoFisher Scientific, Waltham, MA, USA) was diluted in 3% BSA in PBS with NP-40. For loading control, the membrane was probed with mouse monoclonal anti-actin antibody (Cell Signaling, Danvers, MA, USA). After treatment, the membrane was washed and developed by enhanced chemiluminescence using an ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).