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. 2007 Nov 26;204(12):2789–2796. doi: 10.1084/jem.20070607

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Copyright © 2007, The Rockefeller University Press

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Figure 4. — Identification of the antigen recognized by NKM 16–2-4. (A) Immunoprecipitation and Western blot analysis with NKM 16–2-4 were performed with an M cell lysate. 4 major bands (3 bands >250 kD and 1 band of ∼150 kD) were precipitated. A subsequent LC-MS/MS analysis identified the three top bands as maltase glucoamylase and the bottom band as alanyl (membrane) aminopeptidase. (B) Lectin blot analysis performed after immunoprecipitation with NKM 16–2-4 showed that the precipitated antigens were all recognized by UEA-1. (C) mFUT1 and mFUT2 genes were transfected into original CHO cells and CHO-derived mutant lines (Lec1, Lec2, and Lec8 cells) with a pIRES2-EGFP expression system, and the specificity of NKM 16–2-4 and UEA-1 for EGFP-expressing transfectants was analyzed. NKM 16–2-4 and UEA-1 specifically reacted with mFUT1- or mFUT2-expressing original CHO cells. The reactivity of NKM 16–2-4 but not UEA-1 to mFUT1- or mFUT2-expressing Lec2 cells was enhanced compared with that to mFUT1- or mFUT2-expressing CHO cells. On the other hand, mFUT1- or mFUT2-expressing Lec1 or Lec8 cells were not recognized at all by NKM 16–2-4.