Localization in lipid microdomains and association with CD19 are necessary for CD38 signals. (A) Nalm-6 cells were treated with 10 mM MβCD (30 minutes, 37°C) prior to lysis and sucrose gradient centrifugation. Aliquots of the 8 recovered fractions were separated on a 10% SDS-PAGE and immunoblotted with anti-CD38 antibody. The localization of the GM1 ganglioside was checked with a dot blot. Untreated cells were used as the control. Loss of lipid microdomain integrity is followed by redistribution of CD38 molecules (left panel) and by impaired CD38 signaling (right panel), as witnessed by lack of ERK1/2 phosphorylation. (B) CD38-mediated Ca²⁺ fluxes were studied in Nalm-6 cells before and after CD19 silencing. sIgM Ca²⁺ signals were checked as control and were not significantly affected by CD19 siRNA treatment. The black histograms represent cell surface expression of the indicated molecules before and after CD19 siRNA treatment, plotted against an isotype-matched control (open profiles). Percentages refer to the number of positive cells. The Ca²⁺ ionophore A23187 was used to check the efficient loading of the cells.