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0344073827
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note
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Similar results were obtained when chymotrypsin or elastase was used instead of trypsin.
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22
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0344935879
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note
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Sequences of YXXØ motifs in the cytoplasmic domain of rat brain SV2a (accession number Q02563) are as follows: RVQDEYSRRSYSRF (amino acids 36 to 49) and GNFLSCFSPEYRRI (amino acids 434 to 447). Amino acid abbreviations: C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; L, Leu; N, Asn; P, Pro; and V, Val.
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23
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0344073826
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note
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Similar results were obtained if adenosine triphosphate (ATP), which may regulate clathrin-coated pit assembly, was present in the cytosol.
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24
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0344505057
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note
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The YQRL peptide had no effect on the coimmunoprecipitation of AP-2 with other AP-2-binding proteins such as clathrin, AP180, amphiphysin I, or epsin.
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0017342078
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6) were bound to 0.5 ml of poly-D-lysine-coated (5 mg/ml) Affi-prep10 beads (BioRad) for 30 min at 25°C. The bead-attached cells were ruptured by sonication for 15 s, washed with 0.5 M tris-HCl pH 7.0, 1 mM dithiothreitol and then with cytosolic buffer, and finally resuspended in 500 μl of cytosolic buffer.
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Jacobson, B.S.1
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0344935878
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note
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Similar effects of the YQRL peptide were seen if AP-2 recruitment onto plasma membrane lawns was analyzed by indirect immunofluorescence microscopy.
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33
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0344073824
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note
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2 and 1 mM phenylmethylsulfonyl fluoride (PMSF) with 20 μg of GST-fusion protein (at least 85% pure) bound to glutathione Sepharose.
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34
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0345367267
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note
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AP-2 and AP-1 adaptor proteins were prepared by separation of the clathrin coat proteins (3 mg/ml) on a Sepharose CL4B gel filtration column in 0.5 M tris-HCl pH 7.4. The adaptor (AP-2 plus AP-1) and clathrin peaks were pooled and concentrated to 0.5 mg/ml and 1 mg/ml, respectively, and either used directly or frozen in liqid nitrogen and stored at70°C.
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35
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0344935868
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note
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2-cytosol mix for 10 min at 37°C with these reagents before sotubilization in 1% triton X-100 containing cytosolic buffer (27).
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36
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0344935876
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note
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We thank T. F. Martin (University of Wisconsin) for the antiserum to the C2B domain of synaptotagmin, R. Jahn (Max-Planck Institute, Goettingen) for monoclonal antibodies to synaptotagmin I, and members of the De Camilli lab for discussions. Supported by grants from the NIH (NS36252 and CA46128) to P.D.C. and a fellowship from the Human Frontier Science Program to V.H.
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