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After picking of an aliquot of colony of S. pombe on the agar, the yeasts were transferred and cultured in the thiamine-free MM-medium to induce the fusion protein for 24 h at 37 °C. Then the cells were seeded in 96-well microplates along with the test samples in the medium containing 1% DMSO and incubated at 37 °C for further 3 h. Distribution of the GST-NLS-GFP-RevNES-fused protein was monitored with a fluorescence microscope to determine MIC values.
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77949488981
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2O (1:9→2:8, liner gradient, 5 min; 2:8, 5 min; 2:8→8:2, liner gradient, 20 min), flow rate = 1.0 mL/min, detection: UV 220 nm] to determine the remaining amount of the tested sample at the indicated times in triplicate. In this experiment, fetal bovine serum was diluted with RPMI1640 medium to modulate enzymatic activity of esterase.
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9
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77949487707
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note
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20 the primary structure of the transition state was respectively generated. Each transition state was further optimized by the semiempirical PM3 method to calculate the potential energy. The activation energy (E1 and E2) was determined by difference of the potential energy between the stationary state and transition state.
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12
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77949485811
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note
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4+H; 271.0782, found 271.0785.
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77949491414
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2 for 24 h. Transfection of pCG-HA-Rev (plasmid encoding HA-tagged Rev protein) and pCRRE/ΔRev (plasmid encoding Gag protein) plasmids into HeLa cells were performed using PolyFect transfection reagent kit (QIAGEN) for 16 h according to the manufacturer's instructions. After the cells were washed, each solution of tested sample at an appropriate concentration in the medium containing 1% DMSO was inoculated and the whole was incubated at 37 °C for further 12 h. Cells were rinsed with cold D-PBS (-) twice and fixed with 4% formaldehyde/D-PBS (-) for 20 min. Then the cells were defatted with MeOH under shaking for 10 min and washed with cold D-PBS (-) thrice. After treatment with 10% FBS in Dulbecco's MEM medium for 30 min, the samples were incubated with anti-HA antibody (Roche) for 45 min followed by incubation with FITC-labeled anti-mouse IgG antibody (Vector) for 45 min. Localization of the HA-tagged Rev protein in the cells was examined with a fluorescence microscope, then image analysis was conducted by Scion image software (Scion) to determine Rev-export inhibitory activity. In the depicted pictures, several cells free from transfection displayed disperse weak fluorescence due to nonspecific binding of the antibodies.
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