FIGURE 1.

CHIP interacts with hTERT in the cytoplasm. A, GST, GST-CHIP, and GST-KIP were immobilized on glutathione-Sepharose and incubated with exogenously expressed hTERT-HA, followed by immunoblotting with anti-HA antibody. GST fusions were visualized by Coomassie Blue staining. Molecular mass markers are shown in kilodaltons. B, H1299 cells co-transfected with hTERT-HA and CHIP-His were treated with 10 μm MG132 for 2 h and subjected to immunoprecipitation (IP) with either anti-His or anti-HA antibodies, followed by immunoblotting as indicated. C, H1299 cells were subjected to immunoprecipitation with either anti-hTERT or anti-CHIP antibodies, followed by immunoblotting as indicated. IgG antibody was used as a negative control. D, cytoplasmic and nuclear extracts were separately collected from H1299 cells transfected with hTERT-HA and subjected to immunoprecipitation with anti-HA antibody. Duplicate blots were immunolabeled with anti-tubulin (for cytoplasmic fraction) and anti-lamin (for nuclear fraction) antibodies to confirm the absence of the cross-contamination in each fraction. E, H1299 cells were co-transfected with hTERT-HA, along with His-tagged wild-type or mutant CHIP as indicated and treated with 10 μm MG132 for 2 h. Immunoprecipitation was carried out with anti-HA antibody to pull down CHIP-His. F, H1299 cell lysates were incubated with anti-CHIP, anti-dyskerin, or mouse IgG antibodies and incubated with protein G-Sepharose. The supernatants (depleted) and immunoprecipitates (IP) were analyzed by immunoblotting with antibodies against CHIP and dyskerin. G, supernatants and immunoprecipitates were analyzed for telomerase activity by the TRAP assay. To test RNA-dependent extension, RNase A (0.25 mg/ml) was added to the extracts before the primer extension reaction. ITAS represents the internal telomerase assay standard.