Figure 2. Selective dysregulation of Lyn phosphorylation in E613R B cells.

(A) Whole-cell lysates of resting B cells from E613R, +/+, and H/H mice were blotted with Ab to the inhibitory and activating tyrosines of Lyn (Lyn pY507/Src pY416). Src pY416 Ab binds activating tyrosines of all SFKs while Lyn pY507 is relatively specific. Total Lyn, Fyn, and Erk1/2 are detected as loading controls. Data are representative of at least three independent experiments.

(B, C) Fyn (B) and Lyn (C) were immunoprecipitated from whole-cell lysates of resting B cells from E613R, +/+, and H/H mice. IPs were subsequently blotted with Ab to the inhibitory and activating tyrosines of Fyn (B) and Lyn (C). Total Fyn (B) and Lyn (C) are detected as loading controls.

Data in 2B, C are representative of at least two independent experiments.

(D) Whole-cell lysates of resting and anti-IgM stimulated B cells from +/+ and E613R mice were blotted with Ab to SHP-1 pY564 and SHIP1 pY1020 along with total SHP-1 and total SHIP1.

(E, F) BCR-stimulated, fixed, and permeabilized lymphocytes were stained for phospho-Erk (E) or phospho-S6 Kinase (F) and costained for CD23 and B220 so that B cells could be identified. Data were collected by flow cytometry. Histograms depict intracellular phospho-Erk (E) or phospho-S6Kinase (F) in LN B cells from +/+, H/H, and E613R mice. Data are representative of at least three independent experiments.

(G) CD45.1+ BJ, E613R, and H/H lymphocytes were loaded with Indo-1 dye, stained for CD23, and stimulated with 10μg/ml anti-IgM or ionomycin. Ratio-metric assessment of intra-cellular calcium in CD23+ gate was carried out by flow cytometry. Data in 2D, E, F, G are representative of at least three independent experiments.