Figure 1.

Cytotoxic lymphocyte granule-mediated cell death and perforin induce hypercitrullination in target cells expressing PAD2 or PAD4. (A and B) 293T cells expressing PAD2 (lanes 2–5 in A and 1–4 in B) or PAD4 (lanes 7–10 in A and 5–8 in B) were pre-incubated in the absence or presence of inhibitors of granzyme B [compound 6 (Comp-6)] or effector caspases (z-DEVD-FMK), followed by co-incubation in the absence or presence of LAK cells (A) or YT granule contents (GC) (B). In A, LAK cells were incubated alone as controls (lanes 1 and 6). After terminating the reactions, the samples were analyzed by immunoblotting using anti-PAD2, anti-PAD4 (upper panel), and anti-PARP (middle panel) antibodies. Global citrullination was detected by anti-modified citrulline (AMC) immunoblotting (lower panel). Filled and unfilled arrows denote intact PARP and its apoptotic fragment, respectively. (C) 293T cells expressing PAD2 or PAD4 were incubated in the presence of increasing amounts of human purified perforin. The lytic activity of each perforin concentration is indicated. After terminating the reactions, the samples were electrophoresed and the proteins were visualized by immunoblotting using AMC, anti-PAD2 (left panel) or anti-PAD4 (right panel) antibodies. The experiments were performed on at least two separate occasions, with similar results.