Membrane localization of CD38 molecules in human tonsillar B cells. (A) Cell surface expression of CD38 (open profile) in tonsillar B lymphocytes. Background staining is shown in gray. X-axis: intensity of fluorescence; y-axis: number of events. (B) Tonsillar B cells were lysed in 1% Brij98 and the lysates fractionated into a supernatant of soluble proteins (S) and a pellet (P) of insoluble proteins by centrifugation. Proteins were separated on 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose membranes, and blotted with the anti-CD38 antibody SUN-4B7. Where indicated, ODG was added to the lysis buffer. The data shown are representative of 3 independent experiments. (C) Tonsillar B cells lysed as indicated in the Membrane fractionation paragraph of Materials and methods were fractionated on a sucrose gradient. Aliquots of the 8 recovered fractions were separated on a 10% SDS-PAGE and immunoblotted with the indicated antibody. The localization of the GM1 ganglioside and of CD45 was checked with a dot blot as a control of the correct separation of the different membrane fractions. The data are representative of 5 independent experiments (D) The intensity of the bands corresponding to the CD38 monomer (black histogram) and dimer (open histogram) in each fraction was measured using the ImageJ software. The bars represent the sum of the bands in fractions 2 to 4 (DIM) or 5 to 9 (DSM) divided by the sum of all fractions.