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note
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Bacterial membranes were prepared from late log-phase cultures of E. cloacae (isolated from SPF mice or the reference strain ATCC 29941) as previously described [P. J. Henderson and A. J. Macpherson, Methods Enzymol. 125, 387 (1986)]. They were loaded in a single broad well of an SDS (12%)-polyacrylamide gel in SDS buffer. Proteins were transferred to nitrocellulose and visualized by reversible Ponceau Red staining. The membrane was sectioned into nitrocellulose strips, each with an identical profile of bacterial proteins. After blocking with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), each strip was incubated [1 hour, at room temperature (RT)] with intestinal washing IgA diluted in BSA/PBS at the IgA concentration shown (Fig. 1A), or serum at 1 in 100 dilution (Fig. 1B). Bound immunoglobulin was visualized by peroxidase-conjugated, affinity-purified, goat anti-mouse IgA, IgG, or IgM (Sigma).
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0343019908
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note
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H region. PCR products were purified and subcloned for automated sequencing.
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18
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0343891258
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unpublished data
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A. J. Macpherson et al., unpublished data.
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Macpherson, A.J.1
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20
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0343455501
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note
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2). Cells were washed off, and plates were incubated successively with 1:1000 affinity-purified goat anti-mouse IgA (Sigma) and 1:1000 alkaline phosphatase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch) before development of alkaline phosphatase color reactions.
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21
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0006379753
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The aly/aly mice do contain cryptopatches, which are intestinal lymphopoietic sites for T but not B lymphocytes [H. Saito et al., Science 280, 275 (1998)].
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23
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0343019904
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note
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a (PharMingen) were diluted in 100 μl of PBS containing 3% BSA 1:3 over seven to nine steps starting from 1 in 30, 1 in 3, or 3 μg/ml, respectively. Lower dilutions of serum were used in some experiments to compare overall binding between serum and intestinal washings, although the technique of intestinal washing does inherently dilute the secretory IgA sample. After washing, the secondary antibody was goat affinity-purified, peroxidase-conjugated anti-mouse IgA (Sigma; 1:1000 in 100 μl of PBS containing 3% BSA), and the enzyme reaction was developed. Bacterial protein or LPS binding was measured similarly after coating with 1 μg/ml of pure OmpF protein (24), 5 μg/ml of pure LPS isolated from cecal wild-type E. cloacae [O. Westphal and K. Jann, Adv. Carbohydr. Chem. 25, 83 (1966)] or from E. coli O26B6 (Sigma), 1 μg/ml of purified chimeric Xa-CAT or 1 μg/ml of purified FimH (26).
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24
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0026779245
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S. W. Cowan et al., Nature 358, 727 (1992).
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25
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0343019905
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note
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10 CFU of the transformed bacteria every fourth day for 28 days, and ampicillin was administered with the drinking water.
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26
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0030929465
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0342585651
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unpublished data
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A. J. Macpherson et al., unpublished data.
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Macpherson, A.J.1
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28
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0343455498
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Supplementary Web material is available to Science Online subscribers at www.sciencemag.org/feature/ data/1047914.shl
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29
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0343455497
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note
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lo) and injected in basal salts solution. Chimeras were analyzed after 5 to 6 months.
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32
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0343019903
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note
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/ mice, to P. Sebbel and R. Glockshuber for the purified FimH protein, to I. Bloomfield for E. coli strain AAEC356, to G. Dougan for advice about in vivo expression, to B. Odermatt for immunohistology and to B. Ludewig, K. McCoy, K. Maloy, and A. Lamarre for helpful discussion.
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