HEp-2 reactivity is not dependent on variable region oligomannose glycans. (A) Number of N-glycosylation motifs in heavy (VH) and light (VL) chain variable regions, not encoded by the germline sequence. The number of sequences analyzed is indicated in the center of the pie chart. (B) Tumor Igs from patients 0998 and 0912 were produced in the absence or the presence of tunicamycin to generate Ig lacking N-linked glycans. Tumor Igs were then treated with Endo H or PNGase F to confirm the presence or absence of oligomannose glycans or N-linked glycans, respectively. Proteins were separated by SDS-PAGE and immunoblotted for human IgG. (C) HEp-2 IFA staining patterns of tumor Igs with and without (indicated by a “T”) N-linked glycans. Results are representative of 2 independent experiments. (D) Intracellular flow cytometric titration curve of HEp-2 cells stained with tumor Ig with and without (indicated by a “T”) N-linked glycans. Results are representative of 2 independent experiments.