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Fig. 4. ATR influences the retention time of GFP-Rad9 in damage-induced foci. (A) U2OS cells expressing GFP-Rad9 were transfected with ATR or Luciferase siRNA oligonucleotides, and treated with aphidicolin. Half the nucleus containing GFP-Rad9 foci (see rectangle) was bleached for 2.7 seconds at 100% laser intensity, after which redistribution of fluorescence was monitored by recording images every 60 seconds. Western blot analysis of cell extracts from a representative experiment is illustrated (right panel). (B) Quantification of simultaneous FLIP-FRAP experiment as in A, with the exception that the cells were imaged every 30 seconds. Cells were treated with aphidicolin (left panel) or UV (right panel), after which FLIP was measured in the unbleached part of the cell and FRAP was measured in the bleached part of the cell. Control cells (UNT) are either left untreated or treated with damaging agents but not displaying GFP-Rad9 foci. The mean of the data points of individual cells analysed is illustrated (n=number of cells) with error bars representing the s.e.m. (C) Difference in relative fluorescence in bleached and unbleached parts of the nucleus from B, plotted against time. Error bars represent twice the s.e.m.
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