FIG. 6.

Phosphorylation at tyrosine 707 signals nuclear export of hTERT. (A) 293 cells overexpressing myc-tagged hTERTwt or hTERT(Y707F) were incubated with 500 μM H₂O₂ 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 (PY [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 using an anti-myc antibody. The input lane represents 1/10 of cell lysate used for immunoprecipitation. SN, supernatant of immunoprecipitate. (B) 293 cells overexpressing myc-tagged hTERTwt or hTERT(Y707F) were incubated with H₂O₂ for 2 h. Immunoblotting of the nuclear and cytosolic fractions was performed with an antibody against myc (upper panel), topoisomerase 1 (middle panel), and HSP70 (lower panel). Representative immunoblots are shown (n = 5). (C) 293 cells overexpressing myc-tagged hTERTwt or hTERT(Y707F) were incubated with 500 μM H₂O₂ 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). (D) 293 cells overexpressing myc-tagged hTERTwt or hTERT(Y707F) were incubated with H₂O₂ for 2 h, and telomerase enzyme activity in the nuclear fractions was measured. Densitometric analysis is shown (n = 3). *, P < 0.05 versus hTERTwt plus H₂O₂. (E) Lysates from 293 cells overexpressing myc-tagged hTERTwt or hTERT(Y707F) were incubated with H₂O₂ for 2 h. Cell lysates were immunoprecipitated with an anti-myc antibody, and immunoblot analysis was performed with an anti-Ran antibody (middle panel) and an anti-myc antibody (upper panel). The lower panel shows the quality of the immunoprecipitation using an anti-myc antibody. The input lane represents 1/10 of the cell lysate used for immunoprecipitation.