FIGURE 6.

Full-length PEDF with alterations in Arg⁹⁹ and His¹⁰⁵. Full-length PEDF proteins were expressed using an expression cassette for PEDF with a 3X-FLAG tag fused to its N-terminal end. The recombinant proteins were secreted by BHK cells stably transfected with expression vectors containing the particular mutated SERPINF1 cDNA and purified by a two-step ion exchange column chromatography. A, binding assays by peptide P1 affinity chromatography of each recombinant protein was performed, and input (I), unbound (U), and bound (B) material were resolved by SDS-PAGE followed by Western blot using a specific anti-PEDF antibody. Binding reactions and washes were with 50 mm Tris, pH 7.5, 300 mm NaCl, 0.005% Tween 20. Incubations were at 4 °C for 1 h. B, the protective effects of recombinant 3X-FLAG-PEDF proteins assayed in R28 cells. R28 cells in serum-free media were treated with effectors for 48 h. Cytoprotection was assayed by counting TUNEL-positive nuclei relative to total number of cells counted from (Hoechst stained nuclei). Each bar corresponds to the average of percentage of TUNEL-positive nuclei per total number of cells from duplicate wells per assay ± S.D. The recombinant FLAG-fused PEDF proteins are indicated as Arg⁹⁹, His¹⁰⁵, and WT. SFM indicates no treatment. C, protective effects of recombinant 3X-FLAG-PEDF and 3X-FLAG-PEDF[H105A] proteins in vivo. Proteins were intravitreally injected into rd1 mutant eyes of PN11 mice, and photoreceptor survival was assayed a day later as described in Fig. 4. Plot of percentages of TUNEL-positive photoreceptor nuclei (TUNEL-positive/total number of photoreceptor nuclei) as a function of amount of each protein injected. Each point corresponds to the average of the percentages of TUNEL-positive photoreceptors (TUNEL-positive/total number of photoreceptor nuclei) from at least three eyes ± S.D. For Student's t test: **, p ≤ 0.01.