Ab196739

Retinas and cells were homogenized in a western lysis buffer (30 mM Tris-HCl; pH 7.5, 5 mM EDTA, 1% Triton X-100, 250 mM sucrose, 1 mM sodium vanadate and protease inhibitor cocktail), the lysates were then centrifuged at 14,000 ×g for 15 min at 4 °C, and the supernatant was used for protein estimation and further analysis. Equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 8%–10% gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). To determine the presence of VAP-1 in the vitreous samples, equal volumes (15 μl) of vitreous samples were boiled in Laemmli’s sample buffer (1:1, v/v) under reducing conditions for 10 min and analyzed as described (6,10,11). Immunodetection was performed with the use of the anti-VAP-1 antibody (1:1000, ab196739, Abcam), anti-ICAM-1 antibody (1:500; sc 8439, Santa Cruz, Biotechnology Inc.), and anti-vascular cell adhesion molecule-1 (VCAM-1) antibody (1:500, sc 13,160, Santa Cruz, Biotechnology Inc.). Membranes were stripped and re-probed with an antibody against β-actin to evaluate sample processing and lane loading. Bands were visualized using a high-performance chemiluminescence machine (G: Box Chemi-XX8 from Syngene, Synoptic Ltd. Cambridge, UK), and the intensities were quantified using the GeneTools software (Syngene by Synoptic Ltd.).

HRMECs were cultured in coverslips in six-well plates and starved in minimal media overnight. They were left untreated or were treated with 100 ng/ml of cytokine-HMGB1, lipopolysaccharide free (Cat. No. REHM120, IBL International Corporation) for 24 h and were fixed with ice-cold acetone or 4% paraformaldehyde followed by blocking with 10% goat serum for 1 h. Without being washed, the cells were incubated with anti-HO-1 (1:100, sc-10789, Santa Cruz Biotechnology, Inc.), anti-VAP-1 (1:100, ab196739, Abcam), or anti-8OHdG (1:50, ab62623, Abcam) primary antibodies overnight at 4 °C. After washing, cells were incubated with secondary antibodies conjugated with appropriate fluorophores (Alexa Fluor 488 or 555) diluted in 10% goat serum (1:1000) for 1 h at room temperature. After washing, the cell-containing coverslips were counterstained with DAPI before being mounted on a glass slide with the use of the UltraCruz^TM Mounting Medium (sc-24941, Santa Cruz Biotechnology Inc.). Immunofluorescence microscopy was performed using an Olympus motorized system microscope (BX61, Olympus Corporation, Tokyo, Japan) at 20X or 40X magnification.