Figure 5.

FasL expression in melanosomes purified from melanoma cells. (A) Western blot analysis of purified melanosome preparations. Western blot analysis was performed using proteins derived from melanosomal preparations purified from melanoma lines by sucrose gradient. Staining with G247 mAb revealed the presence in melanosome preparations of the 40–33 kD band pattern (lane 3–5). (B) Purity controls of melanosome preparations. Controls performed to check purity of melanosome preparations revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, mitochondria, or plasma-membrane markers. The ER marker Bip/GRP78 was detectable in melanosome preparations. (C) Cytofluorimetric analysis of FasL expression on purified melanosomes. Melanosomes were stained with anti-FasL NOK-1, anti-gp100, anti LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. Melanosomes were detected as granules of the approximate mean size of 1 μm, relative to standard beads of 6 μm size.