Figure 4.

Reduced ADAM10 processing and expression in Gα_i-deficient transitional B cells. (A) CD23 or ICOSL expression on Gα_i-deficient B cells. Representative flow cytometry patterns of CD23 expression on transitional B cell type 2 (T2) cells from WT (Gnai2^fl/fl) and either Gnai2^fl/flmb1-cre or double knockout (DKO) mice and ICOSL on transitional B cell type 1 (T1) and T2 cells from Gnai2^fl/flvav1-cre mice. (B) Immunoblot to detect ADAM10 expression in lysates from two WT (Gnai2^fl/fl) and two DKO splenic B cells. Experiment repeated twice with equivalent results. The ratio between the unprocessed (*) and mature (**) ADAM10 is show. Actin immunoblot shown to verify equal loading. (C) Flow cytometry showing expression of ADAM10 in bone marrow and in splenic B cell fractions. Experiment was repeated a minimum of three times with a similar result. (D) ADAM10 expression on B220⁺CD93⁺IgM⁺ spleen cells as a function of CD23 or CD81 expression using cells from WT (Gnai2^fl/fl) or DKO mice. Flow cytometry showing a plot of ADAM10 versus CD23 (left) or CD81 (right). The % of positive cells in each quadrant is indicated. (E) pTAOK expression in WT (Gnai2^fl/fl) and DKO splenic B cells. Intracellular flow cytometry assessing phosphorylation of TAOK proteins in splenic B cells from WT and DKO mice. Quantification of pTAOK proteins in transitional B cell type 0 (T0) and T1 B cells as measured by flow cytometry from four WT and four DKO mice. Data are mean ± SEM (*p < 0.05).