Figure 6.

OSF-2 contains an evolutionarily conserved secretion signal. (A) Alignment of predicted secretion sequences in various OSF-2 homologs. Phylogram constructed on the basis of amino acid sequence similarities depicting the evolutionary relationships among OSF-2 proteins of different species. The sequence of the predicted human secretion signal (aa1-21) is conserved in all compared homologs. * Identical residues (dark grey), conserved substitutions/similar characteristic (light grey), semi-conserved substitution/similar shape (white). Organisms and amino acid positions are indicated. (B) OSF-2-GFP transfection in different tumor cell lines revealed a cytoplasmic granular localization. No secretion granulae were observed upon expression of the secretion mutant, ΔSec-GFP. (C) Expression of the signal alone fused to GFP (aa1-21; Sec-GFP) was sufficient for the formation of secretion vesicles. GFP expression served as the negative control. Scale bars, 5 µm. (D) Western blot confirming OSF-2 secretion. (E) Immunoblot analysis of cell fractions from OSF-2-GFP and ΔSec-GFP HEK293T transfectants. Only OSF-GFP was detectable in the membrane fraction of secretion vesicles and the supernatant. In contrast, the OSF-ΔSec-GFP secretion mutant failed to be incorporated into vesicles or to be secreted. Probing with anti-GAPDH (cytoplasm), anti-Vimentin (membrane, cytoskeleton), and anti-Lamin B1 (nuclear) Abs served as controls for lysate preparation. Representative results for n = 2 are shown. The uncropped western blot figures were presented in Figures S11 and S12.