American Society of Hematology

Figure 3.

$Figure 3. Translocation of nucleolin is mediated by VEGF and extracellular matrix. (A) Cell-surface nucleolin of HMECs was determined by FACS as described in “Materials and methods.” For the histograms, NC indicates negative control; isotype Ab, isotype Ab control; ST, serum-starved cells; Matrix, HMECs grown on coated matrix protein-fibronectin; VEGF, HMECs cultured with serum-free medium containing 10 ng/mL VEGF. In HMECs grown on fibronectin, VEGF significantly enhanced the surface expression of nucleolin, but the 2 factors have only a marginal effect. (B) HMECs grown on fibronectin were stimulated by 10 ng/mL VEGF for 0 hours, 1 hour, 6 hours, or 12 hours, and then restarved by serum-free medium (SF). The membrane proteins were biotinylated and pulled down by strepavidin-conjugated agarose; meanwhile, total proteins were from whole-cell lysate. Both fractions were subjected to immunoblotting by antinucleolin Abs. The amount of nucleolin in cell membrane (nucleolin-CM) increased following VEGF stimulation and decreased without stimulation; however, during the process, the amount of nucleolin in whole cells (nucleolin-total) did not change. β-actin was used as a loading control. (C) Cell membrane protein CD31, cytoplasmic protein cytochrome C, and nuclear protein Histone-H1 were detected in both streptavidin pull-down fraction and supernatant, to prove that the streptavidin matrix sharply separates biotinylated from nonbiotinylated proteins, and to exclude the traces of nucleolar proteins. (D) After HMECs were plated on immobilized fibronectin (FN) or poly-L-lysine (PLL), the distribution of nucleolin (green) was visualized by immunofluorescence at different times. Bar, 10 μm. (E) Cell-surface nucleolin (green) and nuclei (blue) in HMECs grown on immobilized FN or PLL were stained. Cells were not permeabilized here to visualize cell surface proteins. Bar, 10 μm. (F) During the translocation of nucleolin, stress fibers and focal adhesion formed simultaneously. Actin filaments were stained by FITC-phalloidin (green) and focal adhesion complexes were visualized by indirect immunostaining of vinculin (green). Nuclei were counterstained with DAPI (blue). Bar, 10 μm.$

Translocation of nucleolin is mediated by VEGF and extracellular matrix. (A) Cell-surface nucleolin of HMECs was determined by FACS as described in “Materials and methods.” For the histograms, NC indicates negative control; isotype Ab, isotype Ab control; ST, serum-starved cells; Matrix, HMECs grown on coated matrix protein-fibronectin; VEGF, HMECs cultured with serum-free medium containing 10 ng/mL VEGF. In HMECs grown on fibronectin, VEGF significantly enhanced the surface expression of nucleolin, but the 2 factors have only a marginal effect. (B) HMECs grown on fibronectin were stimulated by 10 ng/mL VEGF for 0 hours, 1 hour, 6 hours, or 12 hours, and then restarved by serum-free medium (SF). The membrane proteins were biotinylated and pulled down by strepavidin-conjugated agarose; meanwhile, total proteins were from whole-cell lysate. Both fractions were subjected to immunoblotting by antinucleolin Abs. The amount of nucleolin in cell membrane (nucleolin-CM) increased following VEGF stimulation and decreased without stimulation; however, during the process, the amount of nucleolin in whole cells (nucleolin-total) did not change. β-actin was used as a loading control. (C) Cell membrane protein CD31, cytoplasmic protein cytochrome C, and nuclear protein Histone-H1 were detected in both streptavidin pull-down fraction and supernatant, to prove that the streptavidin matrix sharply separates biotinylated from nonbiotinylated proteins, and to exclude the traces of nucleolar proteins. (D) After HMECs were plated on immobilized fibronectin (FN) or poly-L-lysine (PLL), the distribution of nucleolin (green) was visualized by immunofluorescence at different times. Bar, 10 μm. (E) Cell-surface nucleolin (green) and nuclei (blue) in HMECs grown on immobilized FN or PLL were stained. Cells were not permeabilized here to visualize cell surface proteins. Bar, 10 μm. (F) During the translocation of nucleolin, stress fibers and focal adhesion formed simultaneously. Actin filaments were stained by FITC-phalloidin (green) and focal adhesion complexes were visualized by indirect immunostaining of vinculin (green). Nuclei were counterstained with DAPI (blue). Bar, 10 μm.

This Feature Is Available To Subscribers Only