Binding activity of perforin mutants in 293T cells with antibodies. 293T cells were transfected with plasmid(s) expressing wild-type (WT) and/or mutant perforin genes, cell lysates were prepared, and immunoblots were performed using 2d4-perf antibody (top panel). Lysates were immunoprecipitated with perforin antibodies H315 (Santa Cruz Biotechnology, Santa Cruz, CA; second panel), dG9 (Endogen, Woburn, MA; third panel), 2D1 (US Biological, Swampscott, MA; fourth panel) and 2d4-perf (the bottom panel). Immunoprecipitates were immunoblotted under reducing conditions and stained with 2d4-perf antibody. Solid and open arrowheads indicate full-length and truncated forms of perforin, respectively. Arrows correspond to the heavy chain of immunoglobulin (IgH).