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7144249244
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note
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18 column (1,0 × 250 mm; Alltech) and eluted immediately by using a buffer B (0.05% TFA containing acetonitrile) gradient starting at 0% at 10 min to 37, 70, 90, and 100% at 73, 105, 115 and 120 min, respectively, at 50 μl/min.
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17
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7144236309
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unpublished data
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S. Joyce et al., unpublished data.
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Joyce, S.1
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7144224985
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note
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18 and cation-exchange column (1.0 × 250 mm; Alltech) by achieving 15, 60, and 100% buffer B at 15, 105, and 125 min, respectively, at 50 μl/min.
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19
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7144242122
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-
note
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Mass spectra were acquired on a Kratos analytical MALDI-4 mass spectrometer equipped with a curved-field reflectron and a nitrogen laser. About 0.3 to 1.0 μl of each fraction (30 to 40 μl), either directly or after concentrating to∼15 μl, was applied onto the sample probe. Matrix, saturated α-cyano-4-hydroxycinnamic acid in 45% ethanol containing 8.8% formic acid (∼300 nl), was then applied to the sample. A replicate of the sample was similarly spotted, except that, to enhance ion signal, 300 nl of saturated ammonium sulfate was added before matrix (20). Spectra were acquired in positive and negative modes as well as in linear and reflectron modes after application of a 20-kV accelerating voltage. Because different fragments form stable positive and negative ions, when derived from the same molecule, positive and negative spectra provide complementary structural information. The mass analyses reported here are within a mass accuracy of 0.5 dalton.
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21
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7144247603
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-
note
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18 RP-HPLC. Sample elution was initiated after the injection front returned to zero by increasing buffer B concentration to 37, 70, 90, and 100% at 63, 95, 105, and 110 min, respectively, at 50 μl/min.
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24
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7144249243
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note
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3H]Mannose-labeled, sCD1d1 and Db-sol-associated ligands were separated from the heavy and light chains by Microcon-10 (Amicon) filtration to specifically monitor GPI-associated radioactivity.
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26
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7144244783
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note
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3H]PI by Microcon-10 filtration, and radio-activity in the retained solution was measured.
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-
-
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27
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7144246569
-
-
note
-
3H]fucose and rarely to other sugars (33), there are about seven times as many mannoses and fucoses in the heavy chain as in GPI (77). Thus ∼65% of CD1d1 is occupied by GPI before accounting for losses incurred during the purification steps. Assuming 65 to 70% recovery of the ligand [based on peptide recoveries from class I molecules (15, 34)], then >90% of CD1d1 is occupied by GPI.
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28
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0029079055
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7144223890
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b, re-spectiveiy; and A. K. Menon for helpful discussions. Supported by grants from NIH (SJ., S.P.B., and R.J.C.), the Juvenile Diabetes Foundation International (S.J.), the American Cancer Society (S.J.), and the National Research Council-NIH (R.R.B.).
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