Figure 5. Detection of a proteinase K resistant, hydrophobic EBI2 ligand bioactivity in multiple tissues and statin-sensitivity of bioactivity generation by HEK293 cells.

(A) Extracts from the indicated tissues were tested for their ability to attract EBI2-IRES-GFP transduced (GFP+, gray bars) compared to untransduced (GFP-, open bars) M12 cells in the same samples (left graph); control-IRES-GFP vector (CTR) transduced M12 cells were similarly analyzed (right graph). Sp, spleen; Thy, thymus; MP, mouse plasma. Nil indicates medium alone. (B) Migration response of control, PTX or oligomer B (OB, inactive PTX subunit) pretreated EBI2-IRES-GFP transduced M12 cells to spleen extract (Sp). Open bars, GFP- (EBI2-) and gray bars GFP+ (EBI2+) cells. (C) Migration response of transduced M12 cells to proteinase K (PK) treated spleen extract, SDF1 or spleen extract plus SDF1. (D) Migration response of transduced M12 cells to tissue extract fractions eluted from a C18 sep-pak column with the indicated amounts of acetonitrile. Sp, starting spleen extract; FT, flow through. (E) Migration response of transduced M12 cells to culture supernatants from HEK293 cells incubated in the absence or presence of the indicated concentrations of mevastatin. Data in C-E are plotted as in B.