Figure 3.

G184S KI B cells have impaired chemokine receptor signaling and they home poorly to LNs. (A) Intracellular calcium response to near optimal chemokine concentrations using splenic B cells from wild type or G184S KI mice. The indicated chemokine and concentration used are shown. The results shown for each time point is the mean response from duplicate wells seeded with B cells from either wild type or G184S KI mice. Each experimental value is the mean of 2 determinations from 3 wild type and 3 G184S KI reconstituted mice. Assay was done duplicate per mouse. The basal levels of calcium were used to normalize the responses. (B) Peak intracellular calcium plotted as a function of the log concentration of the indicated chemokine concentration. Results from 3 wild type and 3 G184S KI reconstituted mice performed in duplicate. Data shown as mean +/− standard error of the mean. A non-linear regression curve fit generated using Prism software. Statistical differences calculated by one-way Anova. (C) Analysis of Intracellular calcium levels in non-stimulated wild type and G184S KI B cells prepared in from 3 wild type and 3 G184S KI bone marrow reconstituted mice. Data shown as mean +/− standard error of the mean. Statistical differences calculated by unpaired t test. (D) Immunoblotting for pERK, total ERK, pAKT, and total AKT in B cell lysates from B cell stimulated with CXCL12 (100 ng/ml). The B cells were prepared from 2 wild type and 2 G184S KI reconstituted mice. Fold change is shown. (E) Results from a 2 hour homing assay. B cells prepared from 3 wild type and 3 G184S KI bone marrow reconstituted mice were labeled and injected intravenously into 6 wild type mice. The numbers of cells in the blood, spleen, popliteal LN, and the inguinal LNs were determined by flow cytometry. Experiment performed three times with similar results. Data shown as mean +/− standard error of the mean. Statistical differences calculated by t test.