Western blot (WB) was performed according to the previously described methods with some modifications 24 (link),36 (link),41 (link). HREC and RRP were mono- and co-cultured to confluency in 6-well plates and used for WB analysis. In brief, the cells were washed with cold PBS three times, detached with a cell scraper, and collected by centrifugation. The harvested cell pellets were sonicated in cold RIPA buffer containing FAST protease inhibitors (Cat#: S8830, Sigma, St. Louis, MO). The protein concentration was determined with the DC™ Protein Assay kit (Bio-Rad) and Qubit 4 fluorometer.
Before the electrophoretic transfer to 0.45 μm pore-size nitrocellulose membranes, 30–50 μg total protein per lane were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were blocked with 5% non-fat milk (Bio-Rad) or 2% BSA at room temperature for 1 hr and then incubated overnight at 4°C with the following primary antibodies (Table 1 ). After being washed with PBST buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology) for 1hr at room temperature. Signals were developed with enhanced chemiluminescence with a SuperSignal West Pico kit (Thermo-Fisher) and detected with an ImageQuant LAS 500 (GE Healthcare).
Before the electrophoretic transfer to 0.45 μm pore-size nitrocellulose membranes, 30–50 μg total protein per lane were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were blocked with 5% non-fat milk (Bio-Rad) or 2% BSA at room temperature for 1 hr and then incubated overnight at 4°C with the following primary antibodies (
