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5
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85069070252
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-
note
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32P at cycles 1 and 11, predicting the sites threonine-250 (T250) and serine-260 (S260) derived from an incomplete tryptic cleavage. Confirmation of this identification was derived from the generation of phospho-specific T(P)250 and S(P)260 antisera. Both antisera reacted with PKCα, confirming occupation of these sites. The S260 site is not discussed further here.
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9
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0028834864
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C. Y. Dong, P. T. C. So, T. French, E. Gratton, Biophys. J. 69, 2234 (1995).
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(1995)
Biophys. J.
, vol.69
, pp. 2234
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Dong, C.Y.1
So, P.T.C.2
French, T.3
Gratton, E.4
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13
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0029007292
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R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, Y. K. Levine, Anal. Biochem. 227, 302 (1995).
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(1995)
Anal. Biochem.
, vol.227
, pp. 302
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Sanders, R.1
Draaijer, A.2
Gerritsen, H.C.3
Houpt, P.M.4
Levine, Y.K.5
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17
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85069076840
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note
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Following phosphorylation of PKCα, the molecular proximity of MC5-Cy3 and T(P)250-IgG-Cy5 (that is, when bound to the same PKC molecule) leads to FRET from the donor to acceptor fluorophore and a consequent reduction in the fluorescence lifetime of the donor Cy3 molecule. Only the T(P)250-IgG-Cy5 antibodies that are in close molecular proximity to the MC5-Cy3 are detected through the reduction in fluorescence lifetime of the donor Cy3, whereas all the other acceptor T(P)250-IgG-Cy5 antibodies are not detected in the experiment. Therefore, there is no requirement for absolute specificity of the acceptor fluorophore.
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18
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0028900634
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J. A. Goodnight, H. Mischak, W. Kolch, J. F. Mushinski, J. Biol. Chem. 270, 9991 (1995).
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(1995)
J. Biol. Chem.
, vol.270
, pp. 9991
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Goodnight, J.A.1
Mischak, H.2
Kolch, W.3
Mushinski, J.F.4
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19
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85069062475
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A supplementary figure is available at the Science Web site
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A supplementary figure is available at the Science Web site (www.sciencemag.org/feature/data/985808. shl).
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20
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85069066568
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note
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To obtain antibodies to the two putative phosphorylation sites, the following synthetic phosphopeptides were synthesized in the Peptide Synthesis Laboratory (ICRF, London, UK): GSLS(P)FGVS-amide, WDRT(P)TRND-amide, where the S(P) and T(P) denote the phosphorylated residues S260 and T250 respectively. Phosphopeptides were coupled to keyhole limpet hemocyanin using glutaraldehyde, and the conjugate was employed to immunize rabbits. For protein immunoblotting of immobilized proteins, Tris-buffered saline (pH 7.5) was used in place of phosphate-buffered saline. Antibodies were employed at 1:2000 dilution for 1 hour at room temperature or overnight at 4°C. Immunoreaction was detected with ECL (Amersham) according to recommended procedures.
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23
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85069063956
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note
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We fixed and permeabilized 293T cells in methanol at -20°C for 4 min. Primary antibodies were diluted 1:200 in 10 mM tris-buffered saline containing 1% bovine serum albumin, except for MC5-Cy3, which was used at 1:50 dilution. The secondary conjugates used were Cy2-conjugated donkey anti-mouse IgG (1:200) and Cy3-conjugated donkey anti-rabbit IgG (1:400) (Jackson ImmunoResearch Laboratories, West Grove, PA). Confocal images were acquired on a confocal laser scanning microscope (model LSM410, Carl Zeiss Inc.) equipped with a ×63/ 1.4Plan-APOCHROMAT oil immersion objective. Each image represents a 2D projection of sections in the Z series, taken across the depth of the cell at 0.5-μm intervals.
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24
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0001385205
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J. E. Celis, Ed. Academic Press, New York
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P. I. H. Bastiaens and T. M. Jovin, in Cell Biology: A Laboratory Handbook, J. E. Celis, Ed. (Academic Press, New York, 1998), pp. 136-146.
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(1998)
Cell Biology: A Laboratory Handbook
, pp. 136-146
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Bastiaens, P.I.H.1
Jovin, T.M.2
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27
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85069073578
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note
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We thank G. Warren and D. Shima (ICRF) for their valuable comments and for antibodies to p62, galactosyl transferase, p115, and giantin. We also thank M. Zerial (European Molecular Biology Laboratory) for a polyclonal antiserum to Rab7. We are grateful to D. Pappin and H. Hansen for protein sequencing.
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