Fig. 3.

ERK1/2 mediated phosphorylation is involved in nuclear translocation of hS3 in response to hydrogen peroxide treatment. (A) Cells were pre-treated with vehicle alone (lanes 1–4), 25 μM UO126 (lanes 5–8), 10 nM Staurosporine (lanes 9–12) or 10 μM DRB (lanes 13–16) for 1 h, and then exposed to 0.125 mM hydrogen peroxide. At 24 h post hydrogen peroxide treatment, cells were fractionated into cytosol (C) and nuclei (N), and hS3 levels in the fractions were determined by the western blot analysis and densitometry tracing of protein bands. Human S3 levels in sub-cellular fractions are expressed as a percent of total cellular hS3, which is a sum of nuclear and cytosolic hS3 in each sample. The blots were stripped and re-probed with anti-PARP antibody in order to detect the presence of this nuclear marker. (B) Whole cell lysates were prepared from serum starved HEK 293 cells and phospho-ERK1/2 was immunoprecipitated using an anti-phospho-ERK1/2 antibody. The resulting immunoprecipitate was subsequently used to phosphorylate purified GST-hS3 protein by immunocomplex kinase reaction. Supernatant from the kinase reaction was subjected to SDS-PAGE and autoradiography. Top panel demonstrates the immunoprecipitation of phospho-ERK1/2 (detected by western blot analysis using the anti-phospho-ERK1/2 antibody). The bottom panel shows the autoradiogram demonstrating the phosphorylation of hS3. Kinase reactions digested with λ-phosphatase (lane 2, bottom panel) and reactions carried out in the absence of ERK1/2 immunocomplex (lane 3, bottom panel) were also included on the gel. (C) HEK 293 cells were transiently transfected with GFP-hS3, GFP-hS3-T42D or GFP-hS3-T42A, and then treated with vehicle alone or 0.25 mM hydrogen peroxide for 24 h. Sub-cellular localization of hS3, hS3-T42D, and GFP-hS3-T42A was traced by fluorescence microscopy using FITC filter.