Expression of human perforin in murine CTLs. Murine splenocytes from T-cell receptor transgenic, perforin-deficient mice (P14.Prf1^−/−) were transduced with bi-cistronic retroviruses containing GFP and perforin cDNA after stimulation with cognate antigen, as described in “Retroviral transduction.” (A) Flow cytometric detection of GFP and perforin after transduction. These cells were not sorted as transduction was uniform and efficient. (B) Western blot of WT and mutant perforins expressed in murine CTL. Three different monoclonal antibodies were used (P1-8, 2d4, and Pf-344). The precursor and mature isoforms seen in the WT perforin are marked by arrows. The lysates were generated under nonreducing conditions. (C) Detection of degranulated perforin from murine CTLs. Degranulation of splenocytes was achieved by coincubation of antigen-presenting, target cells (gp33-labeled EL4) with transduced splenocytes at an effector/target ratio of 5:1 for 2 hours. For a negative control, effectors were coincubated with EL4 target cells without gp33 peptide labeling. The degranulated perforin was “captured” and immunoprecipitated from the supernatant by δG9 antiperforin antibody as described in Figure 3.