FIGURE 1:

The HaloTag on TERT has no effect on telomerase properties except for a reduction in processivity. (A) Western blot of telomerase immunopurified from HEK293T cells overexpressing various untagged and tagged TERT proteins and TR, probed with an anti-TERT antibody. (B) Northern blot of RNA extracted from immunopurified telomerase variants, probed with three TR probes. Standards are in vitro–transcribed full-length TR and were used to quantify the amount of TR in IPs (values below the lanes). (C) Direct telomerase extension assay at 50 mM KCl of various immunopurified telomerase variants. LC1, LC2, and LC3, labeled DNA loading controls. (D) Quantification of telomerase activity normalized to the loading controls and the number of cells used as input for immunopurification (n = 6, mean ± SD). (E) Quantification of telomerase activity normalized to the loading controls and the TERT level (see panel A, n = 6, mean ± SD, t test). (F) Quantification of telomerase processivity using the decay method (n = 5, mean ± SD, t test). (G) Direct telomerase extension assay at 150 mM KCl (to limit processivity) of 3xFLAG- and 3xFLAG-HaloTag-telomerase immunopurified from HEK293T cells using anti-FLAG resin in the absence and presence of POT1/TPP1. LC, labeled DNA loading control. (H) Quantification of 3xFLAG- and 3xFLAG-HaloTag-telomerase processivity in the absence and presence of POT1/TPP1 using the decay method (n = 5, mean ± SD, t test).