(A) Specificity of mAb YKH1 was determined by Western blot using the unbound fraction (norFN), and the bound and eluted fraction (onfFN) from FDC6-column. Aliquots (0.1 µg protein) of each FN were analyzed with mAbs EP5, FDC6, and YKH1. (B) Separation of norFN and onfFN by immunoaffinity absorption. Aliquots (0.1 µg protein) of tFN, norFN (unbound to FDC6) and onfFN (unbound to YKH1) were analyzed by Western blot using EP5, YKH1 and FDC6. (C) Production and secretion of onfFN in HUH7 cells overexpressing GalNAc-T6 (HUH7/T6). Expression of GalNAc-T6, total FN, norFN and onfFN were detected with mAbs 2E11, EP5, YKH1 and FDC6, respectively, using cell lysates and culture supernatants.