Figure 1. Effect of R28C mutation on perforin function. The gel panel shows a western blot for perforin levels in transiently-transfected RBL cells (A). Results of representative cytotoxicity assays are shown for a RBL cytotoxicity assay (B), and an assay using CD8⁺ T cells from Prf1^−/−.OT-1 mice (C). Data points represent the mean values from 3 experiments ± standard error of the mean. In all of these experiments, effector cells were electroporated with human wild-type or R28C mutant PRF1 cloned into pIRES2-eGFP expression vector. Transfected cells were isolated on the basis of identical eGFP fluorescence and their cytotoxic activity against 51Cr labeled and trinitrobenzosulfonic acid-labeled Jurkat cells (in the case of RBL effectors) or SIINFEKL (ova) peptide pulsed EL-4 target cells (in the case of CD8⁺ Tcells), were tested as described in Materials and Methods.