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Fig. 4. FRAP analysis of STAT3-YFP mobility in the nucleus. HepG2 cells were transfected with STAT3-YFP and living cells were stimulated with 20 ng/ml IL-6. (A) 15 minutes after stimulation, a single STAT3-YFP nuclear body was bleached using the 514 nm laser of the laser-scanning microscope. Subsequently, images were taken at the timepoints indicated. Bar, 10 µm. (B) For quantitative FRAP analysis, ROIs of 1 µm in diameter within the nuclei of STAT3-YFP-transfected HepG2 cells before (blue graph) and 15 minutes after IL-6 stimulation (green graph) were bleached and subsequently the recovery of fluorescence was monitored. Fluorescence before bleaching was normalized to 100. YFP was transfected into HepG2 cells as a control for a freely mobile protein. Unstimulated cells were analyzed as described above (black graph). Each graph represents the mean values of five independent experiments.
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