Effects of rK5 on endothelial cells are mediated via Beclin 1/Bcl-2 interactions. (A) Representative Western blot analysis for Beclin 1, Bcl-2, and β-actin in rK5-treated HUVECs. Total-cell lysates from rK5-treated HUVECs were collected at different time points and subjected to Western blot analysis. (B) Densitometric analysis of Beclin 1 and Bcl-2 (■) levels. Values were normalized against β-actin and presented as fold increase compared with the basal level (0 hours of treatment). Data represent means of 3 independent blots. Error bars denote SE (*P < .05). (C) Coimmunoprecipitation of Beclin 1 and Bcl-2 in HUVECs. Cells were grown in 5% FBS in M199 medium and supplemented with 40 ng/mL VEGF in the presence of 1.5 μg/mL rK5. Cell lysates from rK5-treated HUVECs were collected at different time points. The lysates (200 μg per sample) were incubated with anti–Beclin 1 antibodies covalently coupled to protein A/G Plus–Sepharose. The immunoprecipitates were analyzed for Beclin 1 and Bcl-2 by immunoblotting. (D) Densitometric analysis of Bcl-2 (□) and Beclin 1 (■) levels. Coimmunoprecipitation experiments were repeated 3 times. Values are presented as fold increase compared with the basal level (0 hours of treatment; *P < .05). Data are shown as means ± SE. (E) HUVECs were treated with 1.5 μg/mL rK5 in the presence of 25 μM zVAD-fmk. Cells were labeled with 0.05 mM MDC (red) and 1 μM Mitotracker Green. Images were recorded using confocal microscopy at 600× magnification and processed as described previously to quantify the number of autophagy vesicles per cell. (F) Values of MDC⁺ vesicles in rK5-treated HUVECs in the presence or absence of 25 μM zVAD-fmk. Data represent values from 2 independent experiments using triplicate cultures; bars denote SE.