Figure 3

NCL is playing a role in the nuclear translocation of PTN. A. Cellular localization of PTN protein in paraffin sections of chicken embryo CAM at day 9 of embryo development. Arrows on the left picture indicate an intense PTN immunoreactivity in the nucleus of endothelial cells (40× magnification). The picture on the right shows a CAM paraffin section treated only with the secondary antibody (20× magnification). B. Equal amounts of protein extracts of chicken embryo CAM from days 6 (d6) and 9 (d9) of embryo development were subjected to immunoprecipitation for IgG or NCL. The precipitated proteins were analyzed by Western blot for the presence of PTN. C. Equal amounts of protein extracts of HUVEC were subjected to immunoprecipitation for IgG, PTN or NCL. The precipitated proteins were analyzed by SDS-PAGE, followed by Western blot analysis for the presence of NCL or PTN respectively. D. Representative immunofluorescent images of HUVEC stained for PTN (red), NCL (green) and nucleus (blue). E. HUVEC lysates or fixed cells after treatment for 24 h with siRNA for NCL were analyzed for the presence of NCL, by Western blot or immunofluorescence respectively. F. Representative immunofluorescent images of HUVEC stained for PTN (green) and nucleus (blue) after down-regulation of NCL expression by siRNA. siNEG, cells transfected with a negative control siRNA; siNCL, cells transfected with siRNA for NCL.