The effects of L557P and K1050E on telomerase activity. (A) Western blot analyses of HEK293T cell lysates expressing the indicated hTERT variants (top) and the corresponding samples purified by IP (bottom). Blots were probed with an anti-FLAG antibody to detect exogenous FLAG-tagged hTERT. (B) Direct activity assay showing extension of a telomeric DNA primer in vitro in the presence of radiolabeled ³²P-dGTP, for WT telomerase and the indicated variants, expressed individually or together. The number of nucleotides added to the primer is shown on the left. LC: ³²P-labeled 30-mer oligonucleotide included as a control for recovery and loading. (C) Quantitation of total telomerase activity relative to WT for the indicated variants or combinations. Data are the mean ± standard error of the mean (SEM; n = 4). *P = .042; ***P = .0003, by repeated-measures 1-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test. (D) Calculation of the amount of telomerase processivity⁷⁵  for the indicated hTERT variants and combinations. For each telomere repeat added by telomerase, the logs of the “fraction of products left behind” (ie, dissociated from the enzyme) were plotted against the repeat number, and the plot was fitted by linear regression, with the slope inversely proportional to processivity. Each data point represents the mean ± SEM (n = 6). (E) Processivity values (defined as −0.693/m, where m is the slope of the line) were calculated from the 6 individual experiments shown in panel D. Data are the mean ± SEM (n = 6). ****P < .0001, by repeated measures 1-way ANOVA, followed by Dunnett’s post hoc test. (F) Line graphs of the intensities of bands in the indicated lanes of the gel in panel B. WT and K1050E telomerase, expressed individually or together (top). L557P and K1050E telomerase, expressed individually or together (bottom). Magnification of the boxed regions of the plots, showing repeat 6 and higher (inset).