Telomerase activity analyses of TERT p.K570N and p.T567M mutants. (A) Telomerase TRAP assay of TERT mutants. Two concentrations of lysate (3 ng and 0.6 ng of protein) are analyzed in a 2-step TRAP reaction. Control reactions with CHAPS lysis buffer in place of lysate (buffer only) and a telomerase positive cell lysate (positive) are included. (B) Telomerase direct assay of TERT p.K570N and p.T567M mutant telomerase reconstituted in vivo. Cell lysates (1.5 μg of protein) of 293FT cells transfected with TERC and TERT-WT (lane 1), T567M (lane 2), or K570N (lane 3) are analyzed by direct primer extension assays. Transfections of TERC (lane 4) or TERT-WT (lane 5) alone are included as controls. A ³²P end-labeled 18-mer oligonucleotide is included as the loading control (L.C.). (C) Western blot of ectopic TERT expression. The glyceraldehyde-3-phosphate dehydrogenase protein is used as the internal control. (D) Northern blot analysis of ectopic TERC expression. The endogenous 5S rRNA is probed to ensure equal loadings.