FIG. 5.

Src kinase family-dependent tyrosine phosphorylation plays a role in nuclear export of hTERT. (A) 293 cells overexpressing myc-tagged hTERTwt were incubated with 500 μM H₂O₂ in the presence or absence of 10 μM specific Src kinase family inhibitor PP1 for 15 min. Lysates of nuclear fractions were immunoprecipitated (IP) with an anti-myc antibody, and immunoblot (IB) analysis was performed with an antiphosphotyrosine antibody (upper panel). Immunoprecipitations were controlled by immunoblotting with anti-myc antibody (lower panel). A representative immunoblot is shown. The right panel shows the quality of the immunoprecipitation with an anti-myc antibody. The input lane represents 1/10 of cell lysate used for immunoprecipitation. SN, supernatant of immunoprecipitate. (B) Densitometric analysis of three independent experiments is shown. (C) 293 cells overexpressing myc-tagged hTERTwt were incubated with 500 μM H₂O₂ in the presence or absence of 10 μM specific Src kinase family inhibitor PP1 for 2 h. A representative immunostaining is shown. (Upper panels) Nuclear staining with DAPI (blue). (Middle panels) hTERT-myc staining (red). (Lower panels) Tubulin staining (green). Results are shown from five different transfected dishes. Cells with predominantly nuclear hTERT staining were counted by three independent investigators. The quantification is shown on the right side. (D) Telomerase enzyme activity was measured in nuclear fractions of cells treated with 500 μM H₂O₂ or 500 μM H₂O₂ and 10 μM PP1 for the indicated times. Densitometric analysis is shown (n = 3).