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Blood, Vol. 109, Issue 8, 3342-3350, April 15, 2007
Notch activity synergizes with B-cell–receptor and CD40 signaling to enhance B-cell activation
Blood Thomas et al.
109: 3342
Supplemental materials for: Thomas et al, Vol 109, Issue 8, 3342-3350
Files in this Data Supplement:
- Table S1. Splenic B-cell populations in control and CD19-cre-DNMAML1 mice (PDF, 22.6 KB)
- Figure S1. Phosphorylation analysis of FO B cells (JPG, 371 KB)
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(A) Freshly isolated FO B cells were unstimulated (no treatment NT) or stimulated with anti-IgM for 1 minute and were analyzed for expression of phosphorylated Erk, Akt, or p38. Dashed lines indicate isotype controls for phosphostaining, while solid lines indicate phosphostaining in unstimulated cells (red) or anti-IgMenstimulated (blue) cells. (B) FO B cells were left unstimulated for the indicated times and then analyzed for expression of phosphorylated Erk, Akt, or p38. Dashed lines indicate isotype controls for phosphostaining, while solid lines indicate phosphostaining in cells cultured on OP9-Ctrl (red) or OP9-DL1 (blue) cells. Results are representative of 3 independent experiments. (C) FO B cells were stimulated with anti-IgM (12.5 µg/mL) for the indicated times and were then analyzed for expression of phosphorylated Akt. Lines are as described for panel B. Results are representative of 3 independent experiments. (D) FO B cells were stimulated with anti-CD40 for the indicated times and were then analyzed for expression of phosphorylated Akt. Lines are as described for panel B. Results are representative of 3 independent experiments.
- Figure S2. Analysis of splenic B cells in CD19-cre-DNMAML1 mice (JPG, 170 KB)
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(A) The top tier presents CD21/CD35 and IgM staining on B220+ splenocytes from CD19-cre-DNMAML1 or littermate controls. The bottom tier presents CD23 expression distinguishing MZ precursor B cells (CD23+) and MZ B cells (CD23−). Numbers are the percentage of cells in the gate indicated in parentheses above the plot. Anti-B220 (6B2) was conjugated to APC-Cy5.5, anti-CD21/CD35 (7B6) (BD PharMingen) was conjugated to PE-Cy5.5, anti-IgM (331) was conjugated to PE-Cy7, and anti-CD23–PE was purchased from eBioscience. (B) Histogram analysis of DNMAML1-GFP expression in FO (solid line), MZ precursor (dotted line), and MZ (dashed line) B cells from a CD19-cre-DNMAML1 mouse. Results are representative of 3 independent experiments.
- Figure S3. Experimental controls for T-dependent immune responses (JPG, 291 KB)
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(A) C57BL/6 mice were immunized intraperitoneally with alum alone or alum+50 µg NP-CGG in 200 µL volume, and splenocytes were analyzed 7 days later. Numbers are the percentage of cells in the gate indicated in parentheses above the plot. Lin is anti-CD3–PE-Cy7 (145-2C11) and anti-GR-1–PE-Cy7 (RB6-8C5) staining. (B) Numbers represent the percentage of cells in the gate indicated in parentheses above the plot but from a sample stained separately from that presented in panel A.
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