Fig. 3

NRP2 induced F-actin polymerization via the active actin-binding protein cofilin. a Immunofluorescence analysis was performed using FITC-labelled phalloidin (F-actin; green), and the nuclei were stained with DAPI (blue). An overlay of the two fluorescent signals is shown (× 1000). b F-actin and G-actin fractions were prepared from HUVECs transfected with empty vector or NRP2 overexpression plasmid (Top). HUVECs were treated with either F-actin depolymerization factor (cytochalasin D) as a positive control or F-actin enhancing factor (phalloidin) as a negative control. The F-actin and G-actin fractions were prepared and subjected to Western blot analysis [S, supernatant fraction (G-actin); P, pellet fraction (F-actin)] (bottom). c HUVECs were transfected with si-control or si-NRP2 and subjected to Western blot analysis using the indicated antibodies. d After HUVECs were transfected with empty vector or an NRP2 overexpression plasmid, they were lysed and subjected to Western blot analysis with the indicated antibodies. e HUVEC-vector and HUVEC-NRP2 cells were coimmunostained with FITC-labelled F-actin and antibodies targeting total and phosphorylated cofilin. The fluorescent signals of cofilin or p-cofilin (red) along with F-actin (blue) are shown (× 1000). f HUVEC-vector and HUVEC-NRP2 cells were lysed using cytosol buffer and subjected to Western blotting with the indicated antibodies. g After HUVECs transfected with empty vector or an NRP2 overexpression plasmid were lysed in Triton X-100 buffer, the insoluble and soluble fractions were subjected to Western blotting. h After NRP2 was knocked down, HUVEC lysates were subjected to Western blot analysis with the indicated antibodies