Fig. 1.

Nuclear translocation of hS3 in HEK 293 cells exposed to hydrogen peroxide. HEK 293 cells were exposed to various concentrations of hydrogen peroxide for 24 h (A) or to 0.125 mM hydrogen peroxide for various periods of time (B). Cytosolic (C) and nuclear (N) fractions were prepared by sub-cellular fractionation technique, as explained under Experimental Procedures. A 10 μg aliquot of protein extract from each sample was immobilized by SDS-PAGE and electrotransfer techniques. Subcellular localization of hS3 was detected by immunoblot analysis utilizing a monoclonal anti-hS3 antibody. The same blots were stripped and re-probed with antibodies against PARP (nuclear marker) and NPC₆₂ (nuclear envelope marker), in order to exclude the cross-contamination of the subcellular fractions during sample processing. Human S3 levels in cytosolic and nuclear fractions are shown as percent of total cellular hS3 protein, which was determined by summing the integrated density values (IDV) of cytosolic and nuclear bands from each sample.