Detection of anti-Ge-1 and anti-GW182 antibodies in human reference sera by indirect immunofluorescence. (Panel I) GFP-Ge-1 was expressed in Hep-2 cells and cells were stained with anti-GFP antiserum (a,d,g) and human serum Ge (b), 18033 (e), or IC-6 (h). The human sera stained larger and more numerous P-bodies in cells expressing GFP-Ge-1 compared with nontransfected cells, indicating the presence of anti-Ge-1 antibodies in all three sera. (Panel II) GFP-GW182 was expressed in Hep-2 cells and cells were stained with anti-GFP antiserum (a,d,g) and human reference sera. The number of P-bodies recognized by GW182 reference serum 18033 (b) was increased in transfected, compared with nontransfected cells, indicating the presence of anti-GW182 antibodies. Reference sera Ge (e) and IC-6 (h) stained P-bodies (indicated by arrows) in transfected cells weakly (in the case of Ge) or not at all (IC-6), indicating that these sera contained little or no anti-GW182 antibodies. DAPI staining (c,f,i, in Panels I and II) indicates the location of cell nuclei.

(Continued on next page)

(Panel I) Detection of anti-Ge-1 and anti-GW182 antibodies in human reference sera by immunoblot. Extracts were prepared from Hep-2 cells transfected with GFP-Ge-1, GFP-GW182, or GFP (control) as indicated. Reference sera Ge, 18,033, and IC-6 reacted with GFP-Ge-1, as well as endogenous Ge-1 in Hep-2 cells. Note that rabbit anti-GW182 did not react with a ∼180-kD band, consistent with the absence of detectable, endogenous GW182 in these cells. Serum 18033, but not Ge or IC-6, reacted with GFP-GW182. (Panel II) GW182, but not Ge-1, recruits Ago2 to P-bodies. Hep-2 cells were transfected with either myc-Ago2 and GFP-GW182 (a–c) or myc-Ago2 and GFP-Ge-1 (d–f) and cells were stained with anti-myc and anti-GFP antiserum. DAPI staining in c and f indicates the location of cell nuclei.