Protein Expression Analysis in KGN Cells

Protein isolation and Western blotting were performed as previously described [20 (link)]. KGN cells were lysed using RIPA buffer plus protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, USA). A total of 7 µg (NOX4) or 20 µg (cleaved caspase 3, clCASP3) protein per lane was loaded on a 10% (NOX4) or 12% (clCASP3) SDS gel and run (NOX4: 20 min at 100 V + 70 min at 120 V; clCASP3: 20 min at 75 V + 40 min at 150 V). After blotting (NOX4: 55 min at 100 V; clCASP3: 60 min at 100 V) and blocking with 5% non-fat dry milk (Roth, Karlsruhe, Germany) in Tris-buffered saline with Tween 20 (5 mM Tris, 100 mM NaCl, 0.05% Tween 20, pH 7.5), rabbit anti-NOX4 polyclonal antiserum (1:1000, #7927, ProSci, Fort Collins, CO, USA) or rabbit anti-clCASP3 monoclonal antibody (1:1000, #9664, Cell Signaling Technology, Danvers, MA, USA) were administered to detect these proteins. As a loading control, mouse anti-β-actin monoclonal antibody (1:10000, #A5441, Sigma-Aldrich) was used. HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson ImmunoResearch Europe, Cambridgeshire, UK) were used to visualize specific binding. Band intensities were determined using the FIJI software [21 (link)].