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Fig. 6. Confocal microscopy images of the intranuclear distribution of phosphorylated histone H3 and GFP-MSK1. (A, B) Quiescent wild-type (A) or GFP-MSK1-overexpressing (B) cells were treated with 100 nM TPA for 20 minutes (central panels), 25 ng/ml sAn for 45 minutes (right panels) or left untreated (left panels). Cells were processed for immunofluorescence as described in Materials and Methods. Coverslips were incubated with rabbit anti-GFP (panels i-iii) and rat anti-phosphoS28-H3 (panels iv-vi) primary antibodies. Secondary antibodies used were anti-rabbit-IgG-Alexa-488 and anti-rat-IgG-Cy3 conjugates. Nuclei were examined by laser scanning confocal microscopy for Alexa-488/GFP- (green) and Cy3- (red)s fluorescence. Merged images are presented (panels vii-ix). Scale bar, 4 µm. (C) Quiescent wild-type (panels i-iii) or GFP-MSK1-overexpressing cells (panels iv-ix) were treated with 100 nM TPA for 20 minutes (central panels), 25 ng/ml sAn for 45 minutes (right panels) or left untreated (left panels). Cells were processed for immunofluorescence as described in Materials and Methods. Primary antibodies used were anti-phosphoS10-H3 (panels i-vi) or anti-GFP (panels vii-ix). Secondary antibodies were either Cy3-(panels i-vi) or Alexa-488-(panels vii-ix) conjugated anti-rabbit-IgG. Nuclei were identified by the weak GFP-fluorescence and examined by laser scanning confocal microscopy for Cy3-fluorescence. Scale bar, 4 µm.

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