Gnaq^−/− B cells are more resistant to BAFF neutralization in vivo and are more sensitive to BAFF stimulation in vitro. (A–D) 50:50 mixed BM chimeras were generated using Gnaq^−/− BM (CD45.2⁺) and WT (CD45.1⁺) BM. 8 wk after reconstitution, chimeric animals were injected i.p. with a blocking Ab to BAFF/BLyS (clone 10F4) or an isotype control Ab. (B) Peripheral blood samples were collected from Ab-treated chimeric animals between 7 and 21 d and flow cytometry was used to identify the percentage of B cells that were of WT or Gnaq^−/− origin. Data are shown as the ratio (±SEM) of Gnaq^−/− to WT B cells (percentage of Gnaq^−/− B cells divided by percentage of WT B cells; n = 5–10 mice). P-values were determined using an unpaired Student’s t test. (C) Spleen cells from Ab-treated chimeras were isolated, enumerated, and analyzed by flow cytometry at 21 d after treatment. The numbers of mature splenic B cells (B220⁺CD24^lo) of WT and Gnaq^−/− origin were determined and are reported as the mean ± SD of five mice/group. The fold depletion caused by BAFF neutralization is shown above the bar graphs. (D) The ratio of Gnaq^−/− to WT B cells in the spleen was calculated (percentage of Gnaq^−/− B cells divided by percentage of WT B cells) and is shown as the mean ± SD. P-values were determined using an unpaired Student’s t test. Data in A–D are representative of three independent experiments. (E) Splenic CD43^neg naive B cells were isolated from Gnaq^−/− and WT chimeras and enriched for CD21^lo FOB and CD21^hi MZB cells. The total or enriched B cell subsets were cultured in BAFF-free media overnight. Live cells were recovered from the cultures and stimulated with recombinant BAFF for 0–10 min. Protein lysates were prepared and analyzed by Western blotting. Phospho-Akt (p-Ser473), total Akt, and phospho-S6 ribosomal protein were detected using phospho- and protein-specific Abs. Data in E is representative of two experiments.