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1642276251
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7
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53449102811
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note
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++H): 806.370 (calcd 806.372).
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9
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53449093288
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note
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50 values.
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10
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53449096789
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note
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2. The viability of HeLa cells was tested using an MTT assay after incubation with inhibitors added at various concentrations for 48 h. Treatment with (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) tetrazolium bromide (MTT) was performed according to the manufacturer's protocol. Absorbance at 570 nm was measured using a plate reader.
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11
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18244406798
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Nat
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Berkers C.R., Verdoes M., Lichtman E., Fiebiger E., Kessler B.M., Anderson K.C., Ploegh H.L., Ovaa H., and Galardy P.J. Nat. Methods 2 (2005) 357
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Anderson, K.C.6
Ploegh, H.L.7
Ovaa, H.8
Galardy, P.J.9
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12
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53449084383
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note
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HeLa cells were mixed with various concentrations of clasto-lactacystin β-lactone and MG132 and incubated for 1 h at 37 °C in serum-free medium. Next, they were mixed with 1.0 μM dansyl-KF33955 and incubated for another hour. To stop the assay, the cells were collected by centrifugation (15,000g, 5 min), treated with denaturing buffer (60 mM Tris-HCl pH 8.8, 5 M urea, 1 M thiourea, 5 mM EDTA, 1% CHAPS, 1% Triton X-100) at 4 °C, and lysed by sonication. The samples were separated by SDS-PAGE and protein bands on the gel were visualized by SYPRO-Red staining (Invitrogen Co.). Images were acquired using a FLA3000 laser scanner (Fuji Photo Film Co., Ltd., Tokyo, Japan). Following transfer onto a PVDF membrane, immunodetection using an anti-dansyl monoclonal antibody (Santa Cruz Biotechnology) was performed. HRP-conjugated anti-rabbit IgG (MBL) was used as the secondary antibody. Signals were visualized using an ECL Plus detection kit (Amersham Biosciences) and an LAS-1000 Plus image analyzer (Fuji Photo Film Co., Ltd.).
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13
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33646841837
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note
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2, 0.5 mM EDTA, 1 mM dithiothreitol, and 1 mM ATP). The cells were permeabilized by the addition of 0.025% digitonin and incubation on ice for 5 min. The cytosol was "squeeze out" by centrifugation for 15 min at 15,000g. The supernatants were used for the next experiments.
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14
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53449096492
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note
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10
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15
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0034964524
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Köhler A., Cascio P., Leggett D.S., Woo K.M., Goldberg A.L., and Finley D. Mol. Cell 7 (2001) 1143
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Smith D.M., Chang S.C., Park S., Finley D., Cheng Y., and Goldberg A.L. Mol. Cell 27 (2007) 731
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0036103598
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