File S1 - Fibrotic Remodeling of the Extracellular Matrix through a Novel (Engineered, Dual-Function) Antibody Reactive to a Cryptic Epitope on the N-Terminal 30 kDa Fragment of Fibronectin

Table S1. Enrichment of phages from Library J showing binding to N terminal 30 kDa Fn fragment during four subsequent biopanning steps. Figure S1. Schematic diagram illustrating the cloning strategy for re-engineering of Fn52 phagemid clone to the RGDS-containing Fn52RGDS clone. Figure S2. Cell ELISA assay data with different scFv antibody clones. The figure represents binding of D407 RPE cells to 17 different scFv antibody clones picked up at random. Two clones in the histogram (red and blue bars) showed maximal binding to RPE cells. One of these, designated scFv Fn52 (corresponding to the red bar) showed reproducible binding to target cells. Figure S3. SDS-PAGE profiles. Purified scFv Fn52 (lane 2; Panel A); the corresponding western blot using anti-His antibody (lane3; Panel A). Panel B represents the SDS-PAGE profile of purified fraction of scFv Fn52RGDS. Lane 1 in both panels shows the indicated molecular weight markers. Figure S4. Western blot images using scFvO27 as primary antibody. Full length fibronectin (lane 1), 70 kDa fragment of fibronectin (lane 2) and the 30 kDa fragment of fibronectin (lane 3) respectively. M represents the molecular weight marker lane. Figure S5. Binding of scFv Fn52 and O27 to D407 RPE cells. Immunocytochemical staining, using the scFv antibodies as primary antibody, followed by mouse anti-c-myc FITC-labeled antibody as secondary antibody. Negative control (minus primary antibody) is also shown. The bottom panels are the corresponding images with the nuclear marker, Hoechst dye. The magnification bar corresponds to 50 µm. Figure S6. Fibronectin in D407 RPE cells. Immunocytochemistry of D407 RPE cells carried out to stain fibronectin in presence of DMEM plus 10ûS, using rabbit anti-fibronectin antibody (Sigma; F3648), followed by anti-rabbit FITC-labeled antibody as secondary antibody. Negative control (minus primary antibody) is also shown. The bottom panels are the corresponding images with the nuclear marker, Hoechst dye. The magnification bar corresponds to 50 µm. Figure S7. Fibronectin in ARPE-19 RPE cells. Immunocytochemistry of ARPE-19 RPE cells stained with rabbit anti-fibronectin antibody (Sigma; F3648), in the absence or presence of three scFv antibodies, O27, Fn52 and Fn52RGDS. FITC-labeled anti-rabbit antibody was used as secondary antibody. DAPI staining was done for nuclear staining. An image with no primary antibody (negative control) has been included. Images were acquired (60×) on an Olympus confocal laser scanning microscope system. (PDF)