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Figure 5 | BMC Immunology

Figure 5

From: Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells

Figure 5

Influence of perforin on binding and uptake of granulysin in DC. L. innocua-challenged DC were incubated for 45 minutes with 2.5 μM granulysin (A), or with 2.5 μM granulysin and 2.5 kU/ml perforin (B) at 37°C, or at 4°C (C and D). As a control, cells were treated with 2.5 μM actinfrag and 2.5 kU/ml perforin (E). After the incubation, the cells were fixed and stained with the anti-His Ab for CLSM. Representative phase contrast (right panels) and immunofluorescence images (left panels) are shown, bar = 8 μm. L. innocua-challenged DC were treated for 45 minutes with 2.5 μM granulysin and indicated concentrations of perforin at 4°C (E) or 37°C (F). After the incubation the DC were washed with PBS or with 10 mM arginine in PBS before lysis with 0.5% Triton-X-100 in PBS. The level of bound or internalized granulysin was analysed by Western blotting. Granulysin was detected with the anti-His mAb. As a reference, cellular actin was detected using an anti-β-actin mAb. Relative intensity of the bands was determined using Image J software. Mean values and SD of four independent experiments are presented.

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