Ab2413

Verteporfin, human transforming growth factor (TGF-β1; T7039), plasminogen and D-Val-Leu-Lys-7-amido-4-methylcoumarin were from SigmaAldrich. OXA-06, PD-184352, SR-11302 were from Tocris. For protein detection specific antibodies against PAI-1 (Abcam Cat# ab66705, RRID:AB_1310540), fibronectin (abcam, ab2413), p-ERK-1/2 (Santa Cruz, E4, sc-7383), SDHA (abcam, ab14715) and histone H3 (abcam, ab1791) were used.

Cells were lysed using lysis buffer containing 50 mM Tris (pH 7.5),150 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40 that was supplemented with protease inhibitors. Western blotting was performed under standard conditions. Blots were repeated and quantified 2–3 times per experiment. Representative images of those repeated experiments are shown. Antibodies used were polyclonal WWOX,^{60 (link)} monoclonal GAPDH (Calbiochem, 6C5, CB1001), polyclonal anti-Fibronectin for immunoblotting (Sigma Aldrich, F3648), polyclonal anti-Fibronectin for immunohistochemistry (Abcam, Ab2413), anti-Myc (Santa Cruz, Sc-40), anti-Flag (Sigma Aldrich, F1804) and polyclonal Smad2/3 (Santa Cruz, sc-8332).

Transwell inserts containing hfRPE cells were rinsed in PBS, fixed for 10 min in 4% paraformaldehyde (PFA) in PBS followed by fixation in 1% glutaraldehyde for 30 min at room temperature. Inserts were cut into small pieces and blocked with 1% BSA for 30 min at RT. Primary antibodies were incubated overnight at 4 °C. Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA). Secondary antibodies labeled with Alexa-488 or Alexa-555 (Life Technologies, Grand Island, NY) were incubated for 1 h at RT. Controls were incubated only with secondary antibody. Sections were mounted with fluoromount G (Electron Microscopy Sciences, Hatfield, PA) and visualized by TCS SP5 II confocal laser scanning microscope (Leica). 90° projections: z-stack was built from images taken every 0.5 µm. Tridimensional 90° projections were performed with ImageJ^{68 (link)}. Quantification of fluorescent signal was performed by converting z-stacks to 8-bit binary images and measuring the integrated intensity with ImageJ^{68 (link)}.