Figure 6

Expression of the F11R protein in inflamed endothelial cells: silencing of the F11R gene in HAEC and HUVEC using F11R siRNA. (a). Immunoblots demonstrate the detection of the F11R protein retained in cells (cell lysates) and released into the media of inflamed HAEC and HUVEC. Both aortic and umbilical vein endothelial cells were transfected with either the control, non-targeting siRNA or by the specific F11R targeting siRNA (as detailed in the Material and Methods section). Subsequently, the cells were treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs, followed by SDS-PAGE and immunoblotting utilizing F11R antibody (arrows point to F11R), and tubulin, as the protein loading control, of 50 kDa. Lanes 1 and 3 depict the F11R protein as detected in cytokine-treated HAEC or HUVEC transfected with the nontargeting siRNA. Lanes 2 and 4 depict the F11R protein as detected in cytokine-treated HAEC and HUVEC transfected with the specific targeting F11R siRNA.(b). Quantitation of immunoblots of the immunostained F11R protein, detected in the cell culture media of HAEC and HUVEC endothelial cells transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, followed by the exposure of transfected HAEC and HUVEC to a combination of the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. The values for F11R were normalized to tubulin levels by dividing the integrated density of the specific band by the integrative density of the tubulin band. ANOVA statistical analysis was performed on the normalized values. All values are the average of three immunoblots ± SEM. (c). Quantitation of the immunostained F11R protein within the cell lysates of HAEC and HUVEC transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, and further treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. F11R-immunostained protein bands were quantified by normalization to tubulin using image J. The F11R values were normalized to tubulin. ANOVA was performed on the normalized value (n = 3). Values depict the mean ± SEM, * p < 0.005.