Figure 7. The effects of IR and cAMP signaling on the subcellular localization of p53.

REH cells were treated with or without forskolin and IR as described in Figure 1. (A) 4 hours after IR, the cells were subjected to immunocytochemistry for the detection of the subcellular localization of p53 by confocal microscopy, and the cells were co-stained with Hoechst for the visualization of nuclei. One representative of three independent experiments is shown. Scale bars = 10μm. (B) Quantification of the p53 fluorescence intensity of cells from three experiments, analyzing at least 30 cells. The data represent the mean +/-SEM, n=30. ^*p<0.05, (paired t test). (C) Subcellular fractionation of REH cells (20×10⁶) was performed as described in Materials and Methods 4 hours after IR. The cytoplasmic and nuclear fractions were each subjected to immunoblot analyses of p53 expression. The numbers indicated below the p53 image represents the p53 signal intensity relative to the CANX signal, normalized to the ratio in untreated (Ctrl) cells. Left panel: One of three representative Western blots. Right panel: Quantification of the p53 signal in Western blots, normalized to the loading control GAPDH. The data represent the mean +/-SEM, n=3. ^*p<0.05 (paired t test).