Fast protease inhibitor

ARPE-19 cells were washed with cold PBS once and sonicated in Buffer A (25mM HEPES, 150mM NaCl, 10mM MgCl₂, 1mM EDTA, 2% Glycerol, pH7.5), supplemented with Sigma FAST Protease Inhibitor. After quantification using the BioRad DC Protein Assay kit, cell lysates were diluted to 5μg/ml and coated onto the 96-well OxiSelect Protein Carbonyl ELISA plate (Cell Biolabs, Inc., San Diego, CA). Protein carbonyls were derivatized by 2, 4-dinitrophenylhydrazine (DNPH) to DNP hydrazone, and then probed with an anti-DNP antibody, followed by an HRP conjugated secondary antibody. The luminescence was measured at 450nm on a Synergy HT plate reader (BioTek, Winooski, VT).

Modification of the Dot immunoblotting assay [36 (link)] was used to detect the extracellular presence of CXCL13. N1 culture media (5 μl) conditioned by 3 days culture with primary mouse RPE cells and 5 μl of culture media without cultured cells (control) were supplemented with FAST protease inhibitors (Cat#: S8830, Sigma) 1:100 and deposited on a dry nitrocellulose membrane (Bio-Rad). Following 15 min incubation at RT, the total protein deposition was visualized by the incubation with Pierce^™ Reversible Protein Stain Kit (24580; Thermo Fisher Scientific, Waltham, MA, USA). The membranes were then washed with PBS and blocked with 2.5% BSA (A7096, Sigma-Aldrich, St Louis, MO, USA) in PBS at RT for 1 h and then incubated overnight at 4 °C with CXCL13 antibody (Table 1). The membrane was washed three times for 5 min with PBS-T (0.05% Triton) and incubated with HRP-conjugated secondary antibody (1:1000; Biorad) for 1 h at RT. Following additional washing, signals were developed with ECL using a Super Signal West Pico Kit (Thermo Fisher Scientific) and detected with ImageQuant LAS 500 (GE Healthcare).

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).

Western Blotting (WB) was performed as previously described with some modifications [44 , 45 (link)]. RCSC were isolated on ice and sonicated in a cold radioimmunoprecipitation assay (RIPA) buffer containing FAST protease inhibitors (Cat#: S8830, Sigma, St. Louis, MO, USA). The protein concentration was determined using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Before the electrophoretic transfer to 0.45 μm pore-size nitrocellulose membranes, 30 μg total protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4–20% polyacrylamide gel). The protein sizes were resolved by running 10 μl of ExcelBand™ 3-color Regular Range Protein Marker (PM2500, SMOBIO Technology, Inc., Hsinchu City, Taiwan). The membranes were blocked with 5% BSA (A7096) at RT for 1 h and then incubated overnight at 4°C with the primary antibod ies (Table 1). After being washed with a PBS-T buffer, the blots were incubated with an HRP-conjugated secondary antibody (1:1000; Bio-Rad Laboratories, Hercules, CA, USA) for 1 h at RT. The signals were developed with enhanced chemiluminescence with a SuperSignal West Pico kit (Thermo Fisher Scientific, Waltham, MA, USA) and detected with an ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA).