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Blood, Vol. 111, Issue 2, 750-760, January 15, 2008
Multiple signaling pathways promote B lymphocyte stimulator–dependent B-cell growth and survival
Blood Woodland et al.
111: 750
Supplemental materials for: Woodland et al
Files in this Data Supplement:
- Figure S1. BLyS-dependent survival and resistance to atrophy is comparably in cultured wild type B cells and B cells from endotoxin unresponsive TLR4−/− donors (JPG, 77.6 KB)
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CD23+ B cells from wild type B6 or TLR 4−/− donors, were cultured in CM for the indicated times without stimulation, with 1ug/ml of E.coli LPS (055:B5) or with 100 ng/ml of rhuBLyS. TLR4−/− mice 87, were made available by Dr. Shizuo Akira, Osaka University, Osaka Japan, and generously provided by Dr.Egil Lien, Univ. of Massachusetts Medical School. (A) Cell survival was determined daily by counting and trypan blue exclusion. (B) Cell size was determined daily on viable cells using a Coulter Z2 size analyzer. Each data point represents the average of 2 independent experiments with 4 (A) or 2 (B) determinations made for each observation within an experiment.
- Figure S2. BlyS-induced phosphorylation of Akt and mTOR effectors (JPG, 69.5 KB)
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To assess the phosphorylation of Akt and mTOR targets B cells were stimulated using plate-bound recombinant BLyS. 5-6 × 106 purified B cells per sample were spun onto 24-well plates prepared by coating with 5 ug/ml monoclonal anti-FLAG M2 antibody (Sigma), washing and blocking with 1% BSA in PBS, followed by the addition of 2 ug/well of FLAG-tagged rhuBLyS, generously provided by Dr. Randolph Noelle, Dartmouth Medical School, an hour prior to final washing and B cell addition. Cells were removed at various time points, lysed in RIPA, extracts separated electrophoretically and analyzed by Western blot with antibodies specific for the indicated proteins.
- Figure S3. Bcl-2 and Bcl-XL proteins in wild type and Pim deficient B cells resist rapamycin or BLyS-mediated modulation (JPG, 67.8 KB)
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Bcl-2 and Bcl-xL proteins were determined daily in electrophoretically separated extracts of purified wild type (A) or Pim 1−/−2−/− deficient B cells (B) freshly isolated (*) or cultured for two days in CM, with or without BLyS (50 ng/ml) and with vehicle or rapamycin (50 nM). Extracts were transferred to nitrocellulose and sequentially probed with Bcl-2, Bcl-XL and actin specific antibodies.
- Figure S4. BLyS-induced modulation of Mcl-1 in long term B cell cultures (JPG, 61 KB)
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Purified B cells ectopically expressing a human Bcl-2 transgene driven by an Ig enhancer and promoter were cultured in CM with and without BLyS (50 ng/ml). At the indicated times B cells were removed, lysed in RIPA, extracts separated electrophoretically and analyzed for Mcl-1 protein by Western blot. Membranes were stripped and probed with antibody to actin to assess sample loading.
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