Figure 5.

LaminB is cleaved during CTL attack even in the presence of CI or bcl-2 overexpression. (a) Mouse peptide-specific CTL were incubated for the indicated times with an equal number of EL4 cells, preincubated with either medium or the cognate peptide. Lamin cleavage products expected for cleavage by GzmB (46 kDa, *) and GzmA (25 kDa, white arrowhead) are detected with Ab against the C terminus of laminB only in targets incubated with antigen. Although there is a background cross-reactive band in the target cells just below the expected 46-kDa caspase/GzmB fragment, the laminB cleavage signal is clearly enhanced in the presence of specific antigenic peptide. Additional minor laminB cleavage products of ≈42 kDa are also produced specifically during CTL attack. When target cells are preincubated with CI, maintained during the CTL attack, the cleavage products are still clearly seen. E and T indicate lysates of just effector CTL or target cells, respectively. (b) ConA-treated HeLa cells or HeLa cells overexpressing bcl-2 were incubated with human CMV-specific CTL and assayed for lamin cleavage by immunoblot and probed with polyclonal anti-laminB Ab or laminA/C. When granule exocytosis is blocked with EGTA, lamins are not cut. In the presence of Ca²⁺, both expected GzmA and caspase/GzmB fragments are detected. Bcl-2 overexpression does not inhibit lamin B cleavage and CI only partly reduces the appearance of the 46-kDa caspase/GzmB product. Bcl-2 and CI inhibit caspase-mediated laminA/C cleavage.