Figure 4.

Lytic function of perforin expressed in RBL-2H3 cells. (A) RBL-2H3 cells were transduced with perforin-expressing retrovirus, GFP sorted, and used in lytic assays against 2,4,6-trinitrobenzenesulphonic acid (TNBS)-treated RBC targets. To achieve cell conjugation, RBL-2H3 cells were coated with anti 2,4-dinitrophenol IgE and mixed with RBCs at various effector/target ratios (E/T). Percentage lysis was a measure of chromium release from targets. The negative control used RBL-2H3 cells expressing a severely truncated perforin cDNA, 50delT. Each sample is performed in quadruplicate to obtain the mean plus or minus SE. *Statistical significance of Y438C compared with WT or 50delT (2-tailed Student t test, P < .01). T435M was not different from 50delT at each E/T ratio. (B) Degranulation of perforin from transduced RBL-2H3 cells was achieved by treating with PMA/ionomycin for 2 hours. Shown are the Western blots from the cell lysates; perforin is detected by P1-8 and isoforms marked by arrows. Actin was probed to control for protein amount. WT indicates wild-type human perforin. (C) Detection of degranulated perforin from RBL-2H3 cells was achieved by PMA/ionomycin treatment for 1 hour in the presence of a capture antibody in the medium, δG9. The perforin/δG9 complex was precipitated with protein A agarose and then run on nondenaturing gels followed by Western blot with P1-8.