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. 2008 Jul 30;3(7):e2844. doi: 10.1371/journal.pone.0002844

Figure 5. Bid is dispensable for endoplasmic reticulum (ER) stress-induced apoptosis.

Figure 5

Control cells and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) where specified for 1 h before treatment with the ER stressors Tunicamycin, Brefeldin A, and Thapsigargin at the indicated concentrations for the indicated times. A) Cell lysates were subjected to Western blotting with a monoclonal KDEL antibody, which detects Grp94 and Grp78, and a monoclonal α-tubulin antibody. B, C, D) Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). E) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3 antibody, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. F) Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl).