FIGURE 2.

Phosphorylation of H2BS32 is a marker of living cells. H2BS32ph is detectable in normal cycling JB6 cells, and the level is increased by mitogen stimulation in serum-starved JB6 cells. A, the specificity of the H2BS32ph antibody was assessed by Western blot using whole cell lysates with the corresponding specific H2BS32ph and nonspecific unmodified H2B blocking peptide. JB6 cells were disrupted in 1× SDS sample buffer and boiled for 10 min at 95 °C. Total soluble proteins were subjected to immunoblot (IB) analysis using specific antibodies to detect H2BS32ph and total H2B. Incubation of lysates with primary antibodies was performed without (−) or with the competing peptide (shown are peptide dilutions) phosphorylated at Ser³² or the unmodified peptide. Extracts identical to those loaded onto the gel for immunoblot (left panel) have also been probed with the H2B antibody (middle panel) as well as stained with Coomassie Blue (right panel) to assure that identical amounts of proteins were loaded in each lane. B, JB6 cells were seeded into two-chamber slides and cultured overnight. After serum starvation for 24 h, the cells were or were not stimulated with 10 ng/ml EGF for 30 min. Expression and subcellular localization of H2BS32ph were determined by immunofluorescence microscopy. JB6 cells were washed, fixed, and incubated with H2BS32ph and RSK2T577ph antibodies or with antibodies preincubated with 5 μg of the H2BS32ph or unmodified H2B blocking peptide. The nuclei were stained with Hoechst before viewing by immunofluorescence microscopy. Colocalization of H2BS32ph and RSK2T577ph occurs in the nucleus of EGF-stimulated JB6 cells. Staining was not apparent in the densely Hoechst-stained region, indicating that colocalization occurs in the euchromatin, where active gene transcription takes place. An enlarged merged image of all three signals from EGF-stimulated JB6 cells is shown (lower right panel). Scale bar, 10 μm. The degree of colocalization of signals was analyzed according to Manders' overlap coefficient. The intensity scattergram (upper right panel) showed a value of 0.8113, indicating a very strong correlation (overlap) between the two images (H2BS32ph and phosphorylated RSK2 in EGF-treated JB6 cells).