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15
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77955417369
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note
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3/MeOH, 20:1) to afford the product as solid (DD-4-11).
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16
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77955417478
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note
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3/MeOH, 20:1) to afford the product as solid (DD-12-19).
-
-
-
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17
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77955429846
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note
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6. Unless otherwise noted, chemical shifts are expressed as ppm downfield from internal tetramethylsilane, or relative ppm from NMR solvent. Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; b, broad; app., apparent), coupling constants, and integration. Mass spectroscopy was carried out on MALDI-TOF and LC-MS instruments.
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18
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77955421560
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note
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3COONa, pH 4.5) and fixed in 4% paraformaldehyde. To study the reversible effect of these molecules in transferrin uptake, cells were preincubated with dynasore and derivatives (80 μM for dynasore, DD-20 and DD-13; 40 μM for DD-6, 20 μM for DD-11) for 30 min at 37 °C and washed with DPBS for various 0 min, 10 min, 30 min. After washing the cells, 20 μg/ml Texas red transferrin was treated to these cells and incubated for 10 min at 37 °C.
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-
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19
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77955426063
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Note
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Images were obtained with an Olympus IX-71 inverted microscope (Olympus Optical, Tokyo, Japan) with a 40×, 1.0 N.A. oil lens using a CoolSNAP-Hq CCD camera (Roper Scientific, Tucson, AZ) driven by MetaMorph imaging software (Universal Imaging Corporation, West Chester, PA) with a Texas red optimized filter set (Omega Optical, Brattleboro, VT). Light from a mercury lamp was shuttered using a VMM1 Uniblitz shutter (Vincent Associates, Rochester, NY). Analysis and quantification of data were performed using MetaMorph software and SigmaPlot 8.0 (Systat Software, Point Richmond, CA), and data were presented as mean ± SE.
-
-
-
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20
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77955425087
-
-
note
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50 by nonlinear regression curve fitting using Graphad Prism 5 software. GTPase activity of 1 μM Drp1 was measured by same method with dynamin I GTPase activity. To identify the effect of dynasore derivatives to Cdc42 GTPase activity, 1 μM Cdc42 (Cytoskeleton) was mixed with 0.5 mM GST tagged human SNX26 RhoGAP domain, and added to prepared ELIPA mixture with 1 mM GTP. The amount of inorganic phosphate released by GTPase activity was measured by using the GTPase Enzyme Linked Inorganic Phosphate Assay in the presence of 0.5 μM GST tagged human TCGAP RhoGAP domain. The amount of inorganic phosphate released by Cdc42 in the presence of 0.1% DMSO (control), 80 μM dynaosre, DD-6 or DD-11 was measured at 360 nm and plotted as a function of time.
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-
-
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21
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77955413810
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note
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M software (Olympus). Laser lines (488 and 561 nm diode lasers) were coupled to the TIRFM condenser through two independent optical fibers. The calculated evanescent depth was ≈150 nm. Cells were typically imaged in two channels by sequential excitation with 0.15 s exposures and detected with back-illuminated Andor iXon887 EMCCD camera (512 × 512, 16-bit; Andor Technologies). Image J program (National Institutes of Health) was used for analysis.
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24
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65249139458
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K. Miyauchi, Y. Kim, O. Latinovic, V. Morozov, and G.B. Melikyan Cell 137 2009 433
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Cell
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Miyauchi, K.1
Kim, Y.2
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Melikyan, G.B.5
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26
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57749173647
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A. Otsuka, T. Abe, M. Watanabe, H. Yagisawa, K. Takei, and H. Yamada Biochem. Biophys. Res. Commun. 378 2009 478
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