Figure 3: GA induces signaling on B cells, upregulates early activation markers, but fails to induce expression of late activation markers. (A) GA induces the phosphorylation of tyrosines in wild-type B lymphocytes. Purified B lymphocytes were incubated with anti-IgM-488 or GA-488 for the indicated time points, immediately fixed and stained for intracellular pTyr. Shown is the flow cytometric analysis of intracellular pTyr on IgM or GA positive B lymphocytes at the indicated time points. (B) GA upregulates CD69, but not CD86 or MHC Class II and blocks anti-IgM induced upregulation of CD86 and MHC Class II on GA positive cells. Purified wild-type B lymphocytes were incubated with GA for 1 hour followed by anti-IgM stimulation for 24 hours (CD69) or 48 hours (CD86 and MHC Class II). Shown is flow cytometric analysis of expression levels of the indicated activation marker on gated GA negative or GA positive populations. The black line represents cells that were not stimulated with anti- IgM, while the red line represents cells that were stimulated with anti-IgM. (C) Graphical representation of the average ± SD gMFI of CD69, CD86 and MHC Class II from the indicated conditions. Data is representative of three individual experiments with similar results. Statistical analysis was performed using a one-way ANOVA with a Bonferroni post-test.