Fig. 9. FasL is targeted directly to the secretory lysosomes in NK cells. FasL endocytosis was studied in the human NK cell line YT. To demonstrate expression of CD63 and FasL in YT, the cells were stained with anti-CD63 (B) or Nok-1 (E) and co-stained with anti-lamp-1 (A,D). C and F show the merge of the two signals, demonstrating co-localisation of CD63 and FasL with the granules. For endocytosis, YT cells were incubated with an anti-CD63 antibody (G-I), Nok-1 (J-L) or an isotype-matched control antibody (data not shown) in complete medium supplemented with the metalloprotease inhibitor BB3013 for 2 hours at 37°C. The cells were fixed in ice-cold methanol and stained with an anti-lamp antibody (G,J). The anti-lamp and endocytosed antibody signals were detected with the relevant Texas-Red- and FITC-conjugated secondary antibodies, respectively. (I,L) The merged signals demonstrate that most endocytosed antibody was located in the Lamp-positive compartment. All samples were analysed by confocal microcopy. Bars, 5 µm (A-F), 10 µm (G-I).