Figure 3

Immunoblotting supports the proteomic analysis and indicates relative changes of various endothelial cell proteins evoked by endorepellin treatment. (A) Close up view of two representative 2-D gels, depicting the decline in calreticulin and β-actin levels. Notice that the major actin isoforms also show reduced staining intensity in response to endorepellin treatment. (B) Immunoblotting of control and endorepellin-treated endothelial cell lysates using an antibody against human calreticulin. Equal amounts of total proteins were loaded. (C) Immunoblot analysis of β-actin levels following treatment with ~200 nM endorepellin for the designated time intervals. (D) The kinetic data for β-actin expression levels were derived from immunoblotting data as presented in C. Essentially HUVEC β-actin expression levels were examined by Western blot following exposure to endorepellin for the indicated time points. β-actin levels were quantified over similar amounts of protein loading and graphed as percent β-actin levels (based upon time point 0) versus exposure to endorepellin treatment (hours). The data were graphed in SigmaPlot v9 and fit by non-linear regression analysis. Each data point represents the mean ± S.E.M. from three experiments. The T1/2 of ~45 min. represents the time to reduce β-actin levels by 50% as compared to the control. (E) Immunoblot analysis of vimentin and prolyl 4-hydroxylase, β subunit protein levels following endorepellin treatment for the indicated time points. GAPDH is shown as loading control. All verification experiments presented in panels C-E utilized new samples of endorepellin-treated HUVEC lysates, separate from those used in proteomic analysis.