Figure 2.
Stretch plus 30 mm NMDA injury produces cytochrome c release, but not caspase-3 activation. A, Immunoblot of the cytoplasmic fraction of injured cells using anti-cytochrome c (top) antibodies. Reblotting with anti-β-actin (middle) antibodies and Ponceau staining of parallel samples (bottom) was performed as control for protein loading. Cytochrome c is released into cytoplasm almost immediately following stretch plus 30 μm NMDA and persists for at least 20 h (n = 3). B, Immunoblot of caspase-3 (both pro- and active/cleaved forms) from whole-cell lysates taken 20 h after the indicated insult. The procaspase-3 form (∼32 kDa) was detectable under all conditions, whereas only cultures treated with staurosporine displayed the active caspase-3 band (∼17 kDa). Data are representative of four experiments. C, Cell lysates taken 20 h after the indicated insults and incubated with Ac-DEVD-AMC show no increases in caspase-3-like proteolytic activity compared with controls (n = 3 experiments). Asterisk, Difference from HEPES buffer-treated controls (*p < 0.05; n = 3). Stauro, Staurosporine (1 μm) controls harvested at 24 h posttreatment in B and C. D, Immunoblot of whole-cell lysates taken 20 h after the indicated insults using anti-3-nitrotyrosine antibodies (n = 4 experiments). SIN-1 (1 mm for 24 h) was used as a positive control.
