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. 2013 Oct 18;21(1):172–184. doi: 10.1038/cdd.2013.143

Figure 1.

Figure 1

Arrestin-2 is uniformly cleaved by caspases during apoptosis induced by TNFα or etoposide. (a) A3KO MEFs exposed to 10 ng/ml of TNFα with 10 μg of CHX (left) or 100 μM etoposide (right) for the indicated times were lysed and analyzed by western blotting. Black arrow, FL arrestin-2; open arrowheads, 1-408 and 1-380 fragments. Small black arrowheads on the left show the positions of Mr standards (kDa) or characteristic cleavage fragments. Arrestin-2 cleavage parallels the activation of caspase-3, -6, and -8, and the cleavage of caspase substrates PARP and BID. Here and in other figures, GAPDH serves as loading control. (b) A3KO MEFs were pre-treated for 2 h with indicated concentrations of pan-caspase inhibitor z-VAD-fmk and then exposed to TNFα/CHX for 12 h (left) or etoposide for 36 h (right). Cell lysates were analyzed by western blotting with F4C1 monoclonal antibody: short exposure (upper blot) shows WT arrestin-2 levels (black arrow) and longer exposure (lower blot) reveals cleavage products (open arrowhead, 1-380). The activation of caspase-3, -6, and -8 and PARP cleavage was determined in parallel. (c) Purified arrestin-2 (75 ng) was incubated for 3 h at 37 °C in 50 μl of caspase cleavage buffer (low salt) in the presence of 1 unit of indicated active human recombinant caspases (Millipore). Initiator caspases-2, -8, -9 and -10 were also assayed in the presence of 1.0 M ammonium citrate (high salt). The products were analyzed by western blotting with F4C1 antibody. Indicated forms of purified arrestin-2 were run as standards. (d) Caspase-8 fl/fl and caspase-8 knockout (KO) MEFs were treated with TNFα/CHX (left) or etoposide (right) for the indicated times. TNFα induces robust activation of caspases-8, -6, and -3 in caspase-8 fl/fl MEFs and appearance of arrestin-2-(1-380) (open arrowhead) after 6–9 h. Caspase-8 knockout MEFs do not show caspase activation or generation of 1-380. On etoposide treatment, both caspase-8 fl/fl and knockout MEFs show similar activation of caspase-3 and -6 and generation of 1-380. See also Supplementary Figure S1