Journal: BioMed Research International
Article Title: Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo
Figure Lengend Snippet: PEDF secretion by immortalized RPE cells in vitro and in vivo . (a) ELISA-based quantification and Western blot analysis of total PEDF secretion by immortalized rat RPE cells in vitro at passages 1, 2, 5, and 10 (grey bars) after SB100X -mediated transfection with the pT2-CMV-PEDF/EGFP transposon plasmid. Note that nontransfected control cells secreted only very small amounts of endogenous PEDF (white bar). Data represent the mean ± SD from 2 independent measurements ( ∗∗∗ p = 0.0001). (b) Western blot analysis of total PEDF extracted from rat control eyes and eyes at 1 day, 1, 2, and 3 weeks after subretinal transplantation of PEDF-transfected rat RPE cells. Double bands indicated the slightly different molecular weights of endogenous (46.5 kDa) and recombinant human PEDF (47.5 kDa), which included a His-tag. Loading of equal protein amounts was confirmed by similar densities of GAPDH protein bands (36 kDa). Note that for these experiments the number of cells transplanted was 1 × 10 5 , since the low sensitivity of the anti-PEDF antibodies required a larger amount of protein. Data represent the mean ± SD from 2 independent measurements. (c) ELISA-based quantification of recombinant PEDF secretion in vivo in lysates of rat eyes at 1 day, 1, 2, 3, and 4 weeks after subretinal transplantation of 1 × 10 4 PEDF-transfected RPE cells, showed a constant level of recombinant human PEDF. For nontransfected control cells recombinant PEDF was not detectable (left bar), because the analysis was carried out with Ni-NTA HisSorb plates. Each bar represents the average data of three injected eyes ( ∗∗∗∗ p
Article Snippet: ELISA-based quantification was performed using the ELISAquant kit for human PEDF (BioProducts MD) and Ni-NTA HisSorb plates (Qiagen) according to the manufacturer's instructions.
Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Plasmid Preparation, Transplantation Assay, Recombinant, Injection