Western Blot Analysis of Retinal and RPE/Choroid Proteins

WB was performed as previously described with some modifications [41 (link)]. Dissected mouse retina or RPE/choroid was sonicated in cold RIPA buffer containing FAST Protease Inhibitor (Sigma). Protein content from the retina or RPE/choroid was quantified using the Bio-Rad DC Protein Assay kit (Hercules, CA). 5–20 μg protein per lane was separated by 4–12% Bis-Tris SDS-PAGE (Life Technologies) and transferred to 0.2 μm pore size nitrocellulose membranes. Membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated overnight at 4°C with the following primary antibodies: anti-Cxcr5 (1:500, Bioss), anti-ZO-1 (1:500, DSHB), anti-TNF-α (1:500, Janssen, PA), anti-GAPDH (1:2500, Abcam), and anti-β-actin (1:2500, Cell Signaling) followed by incubation with horseradish-peroxidase (HRP)-conjugated secondary antibody (1:4000; Cell Signaling) for 1 h at room temperature. Signal was detected by enhanced chemiluminescence (ECL) using SuperSignal West Pico or Femto kit (Thermo Scientific) and GE Healthcare's ImageQuant LAS 4010 Digital Imaging System (Pittsburgh, PA). Densitometry was performed using Image J (NIH, Bethesda, MD).