Differences in IgM surface expression profiles and BCR component glycosylation status between CLL patients and healthy subjects. (A) Flow cytometry analysis of surface IgM staining. Results from 2 patients, one with weakly expressing IgM (CLL 1 in Table 1) and one expressing IgM more strongly (CLL 8 in Table 1), and from a representative healthy subject are shown. Preparations enriched in B cells were subjected to flow cytometry, comparing isotype control staining (bold line) with test mAb staining (thin line). Fluorescence intensities are shown on a logarithmic scale on the x-axis. We report the percentage of positive cells and mean fluorescence intensity of IgM staining. (B) Glycosylation analysis of BCR components. Cell extracts (20 μg) produced by lysis in MBS were incubated at 37°C in the presence (+) or absence (-) of Endo-H and separated by 10% SDS-PAGE, and the resulting protein bands were transferred to nitrocellulose. Filters were probed with mAbs as follows: rabbit anti-μ heavy chain (μ), mouse anti-CD79a (CD79a), or anti-CD79b (CD79b), and immunoreactive bands were detected with an appropriate horseradish peroxidase-linked secondary antibody. Immature glycosylated (dotted arrows) and mature glycosylated (solid arrows) proteins are indicated for each staining. Forms deglycosylated by Endo-H treatment are indicated by #. Molecular masses are indicated in kilodaltons (kDa). The asterisk indicates a nonspecific band at 30 kDa constantly observed with the anti-CD79b probe from Pharmingen.