Cells were transfected as previously, harvested at 72 h, lysed and 150 µg of total protein (lysate) or the same percentage of total medium was treated with Endo F to completion under native conditions, and analyzed using 2DGE followed by WB. (A1–A4) Analysis of transfected cell lysates was used to define six PAcP-related spots (see text). Wt-PAcP (A1), IgG(NH)-PAcP (A2), HSP (A3), Prl(NH)-PAcP (A4). (B1–B4) The spots defined from transfected cell lysates were compared with spots observed in HSP or media. Wt-PAcP (B1), IgG(NH)-PAcP (B2), Prl(NH)-PAcP (B3), HSP (B4), (C) 25 µl of human serum samples and 5 µl of HSP were precleaned, treated with Endo F to completion under native conditions, and analyzed by 2DGE as previously. Clinical histories were (a) Gleason grade 3+3, stage pT2cNXMX, adenocarcinoma; (b) Gleason grade 3+3, stage pT2cNXMX, small acinar adenocarcinoma; (c) Gleason grade 3+3, stage pT2cNXMX, small acinar adenocarcinoma; (d) Gleason grade 5+4, stage pT3N1MX, invasive prostatic adenocarcinoma; (e) Gleason grade 4+5, stage pT3bN1MX, small acinar. (f) Gleason grade 4+5, stage pT3bN1MX, small acinar; (g) normal serum. adenocarcinoma; (f) Gleason grade 4+5, stage pT2cN0MX, small acinar adenocarcinoma; (g) healthy male control serum; HSP as used in previous panels as a reference. See Supplementary Methods for additional details.