Figure 6.

Transgenic over-expression of Bright decreases BCR signalling of normal B cells. (A) Mobilisation of intracellular Ca²⁺ is reduced by Bright over-expression. Wild type and Bright transgenic mice were sacrificed, splenic CD43⁻ B cells were prepared by negative selection, loaded with Indo-1 and then subjected to measurements of intracellular Ca²⁺ using 1 ng (low), 500 ng (medium) or 40 μg α-μ (high) to stimulate 10⁶ cells; Ionomycin, Digitonin, EGTA and Tris were used for internal calibration. (B) Purification of T1, T2, FO and MZ B-cell populations. B cells were prepared from single cell suspensions of transgenic (shown here) and wild-type (not shown) splenocytes. B220⁺ B-cell subpopulations were defined as T1 (CD93⁺ CD23⁻ CD21⁻), T2 (CD93⁺ CD23⁺ CD21⁺), FO (CD93⁻ CD23⁺ CD21⁺) and MZ (CD93⁻ CD23⁻ CD21⁺). (C) Over-expression of Bright inhibits global phosphotyrosine responses of isolated B-cell populations. Each indicated subpopulation (∼10⁶ cells) was stimulated for 5 min using 1 ng α-μ, 1 ng α-CD19 or 1 ng α-μ+1 ng α-CD19. Whole cell lysates (WCL) were prepared from each and then were subjected to SDS–PAGE/western blotting using α-pY, α-Bright and α-Tubulin (loading control) anti-sera. (D) Trafficking of BCR signalling components in and out of lipid rafts is not disturbed in transgenic B-cell populations. The indicated subpopulations (∼2 × 10⁶ cells each) were stimulated for 5 min using 2 ng α-μ, 2 ng α-CD19 or 2 ng α-μ+2 ng α-CD19. The entire preparation of each was then used for the isolation of lipid rafts. Fractions from the gradient centrifugation were collected and divided into two equal parts. One part was analysed directly by SDS–PAGE/western (lipid rafts from ∼10⁶ cells per lane) using the antibodies indicated to serve as an input control for the IP performed with the other half of the RIPA extracted samples (shown in Figure 7B). Here, increased levels of coprecipitated μ, Raftlin and CD19 indicated coalescence of lipid rafts upon BCR engagement (Depoil et al, 2008).