In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP
Figure 1
siRNA-mediated down-regulation of nucleolin. (a)-(b) Confocal section of live HeLa cells stably expressing nucleolin-mCherry (red) and the histone H2B-eGFP (green) visualized before (a) or after nucleolin silencing (b). Scale bars represent 5 μm. H2B-eGFP and nucleolin-mCherry fluorescence intensity along the white lines are presented under the fluorescent images. (c) Western blot analysis of nucleolin in nonfluorescent HeLa cells: untransfected control cells, control cells transfected only with the transfection reagent, cells transfected with siRNA control high GC and siRNA against nucleolin-treated cells (mix siRNA 2 and 4 at 2 nM). Equal loading was verified using antihistone H3 antibody. (d) Quantitative RT-PCR analysis of nucleolin mRNA in nonfluorescent HeLa cells: untransfected control cells, control cells transfected only with the transfection reagent, siRNA against nucleolin-treated cells (mix siRNA 2 and 4 at 2 nM) and siRNA against nucleolin treated cells (mix siRNA 2 and 4 at 20 nM). Data were normalized with the amount of -actin mRNA and the amount of mRNA in control cells.
