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-/- mice used in these experiments resulted from intercrosses between the 129 strain from which the ES cells were derived and strain C57B1/6.
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Mice were immunized intraperitoneally with 100 μg of TNP-KLH in complete Freund's adjuvant (CFA). After 12 days, splenocytes were lysed and immunoprecipitated with the indicated antibodies. Immunoprecipitates were electrophoresed through a 7.5% SDS-polyacrylamide gel electrophoresis gel, transferred to nitrocellulose, incubated, and developed with a rabbit antibody to BCL-6 (5), with the use of the ECL system (Amersham).
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note
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Bone marrow cells, splenocytes, and thymocytes were analyzed by flow cytometry for the following surface proteins: CD4, CD8, CD45R (B220), IgM, CD3, CD24 (HSA), CD43, CD11b (Mac1), Gr-1, CD90 (Thy-1), CD5, IgD, TCR-αβ, CD25, CD23, CD38, CD80 (B7.1), CD86 (B7.2), MHC class II (IA), CD11a (LFA-1), CD19, CD18, CD40 ligand, CTLA-4, and CD-69. No differences between BCL-6 mutant mice and wild-type littermate control mice were observed.
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21
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1842403867
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note
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Mice 5 to 6 weeks old were immunized intraperitoneally with 100 μg of TNP-KLH in CFA. After 12 days, serum was collected for anti-TNP titers, and spleens were processed for immunohistochemistry by being embedded in OCT compound (Tissue-Tek) and frozen. Splenic cryosections were stained with PNA-horseradish peroxidase, biotin-anti-mouse IgD, and streptavidin-alkaline phosphatase according to standard procedures (15). For T cell-independent immunization, mice were injected intraperitoneally with 25 μg of TNP-conjugated Ficoll suspended in phosphate-buffered saline. Serum was collected for anti-TNP titers after 7 days. Anti-TNP titers were assayed from sera by ELISA on TNP-ovalbumin-coated plates with the use of alkaline phosphatase-conjugated, goat anti-mouse Ig antibodies specific for various Ig isotypes (Southern Biotech).
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1842365762
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unpublished observations
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1842374750
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Etectrophoretic mobility-shift assay (EMSA) DNA binding reactions were performed essentially as described (6). The CD23b DMA probe sequence is 5′-GATCAGGGTGAATTTCTAAGAAAGGGACTGGTGT-3′. BCL-6 protein and the luciferase control protein were generated with in vitro translation as described (6). Nuclear extracts were prepared essentially as described (35). For antibody supershift analysis, 3 μg of antibody [anti-BCL-6 (N3) or anti-State (S20); Santa Cruz Biotechnology] or preimmune rabbit serum was used.
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A CD23b luciferase reporter construct was prepared by tandem ligation of three human CD23b BCL-6/Stat6 sites (24) immediately 5′ to a minimal TK promoter (base pairs -81 to +52) in pGL2basic (Promega). NIH 3T3 cells were transfected by the calcium phosphate transfection method using 2 μg of β-galactosidase (β-Gal) expression vector (pSV2Bgal), 5 μg of CD23b reporter plasmid, 10 μg of Stat6 expression plasmid (in the vector pCEV27) or empty pCEV27 vector, and 10 μg of BCL-6 expression vector [pCGN-BCL-6 (6)] or empty pCGN vector control. The day after transfection, cells were incubated for 16 hours in serum-free medium and then treated with 500 U/ml (0.25 ng/ml) of mouse IL-4 for 6 hours. Cell extracts were prepared and analyzed with the Luciferase Assay System (Promega). Luciferase activity was normalized on the basis of β-Gal activity in each extract.
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1842376568
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note
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6 WI-L2-NS cells were transfected with 6 μg of marker expression vector for mouse intercellular adhesion molecule-1 (ICAM) (pmICAM) and 30 μg of expression vector for BCL-6 [pCGN BCL-6 (6)] or empty vector (pCGN) using a BTX electroporator set at 260 V, 950 mF, and 720 ohms. After overnight culture, the transfected cells were stimulated with recombinant human IL-4 (10 ng/ml; Genzyme) for 16 hours and subsequently stained for flow cytometry with mouse anti-human CD23-PE (M-L233, Pharmingen) and hamster anti-mouse CD54/ICAM (3E2, Pharmingen).
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1842291812
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1842288862
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note
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1842293635
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-/- mice; J. Inman for TNP-Ficoll; W. LaRochelle for the State expression vector; P. Caspar and A. Sher for help with the anti-IL-5 ELISA and helpful discussions; B. Paul for recombinant mouse IL-4 and helpful discussions; K. Kelly for the pmICAM expression plasmid; and T. Waldmann, R. Germain, R. Hodes, and H. Morse for helpful discussions. Mice were maintained according to the NIH Office of Animal Care and Use guidelines.
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