FIG. 6.

Uptake and intracellular localization of GrB and the cs mutants. Binding and accumulation of FITC-labeled transferrin (125 nM) or FITC-labeled granzymes (600 nM) in the presence of sublytic perforin. Jurkat cells were exposed to fluoresceinated protein and perforin at 4°C or 37°C for 15 min, washed, and analyzed by FACS. Autofluorescence levels of cells treated with perforin alone at 4°C (shaded histogram) or 37°C (black line) were identical. Cells from the same populations were also analyzed by CLSM. (A) Uptake and localization of wt GrB compared to transferrin. Image shows the distribution of fluoresceinated protein (FITC) with a corresponding differential interference contrast image. Shown are single optical sections from single cells (left and center panels) or z-stacks of multiple cells (right panels). (B) Uptake and localization of cs mutants at 4°C and 37°C was analyzed as described above. Images show single optical sections. (C) A fluorograph and an immunoblot probed with anti-GrB monoclonal 2C5 demonstrate integrity and equivalent FITC labeling of wt GrB and the cs mutants. The fluorograph was taken prior to transfer to a membrane for immunoblotting (Tf, transferrin).