Fig. 2

Expression, purification and activity of the FLAG-SNAP-TERT protein. a Western blot of lysates of parental HEK 293 cells and edited clones expressing FLAG-SNAP-TERT using a FLAG antibody. Bands at the expected size of FLAG-SNAP-TERT (arrow) indicate that the fusion protein is expressed in the edited clones. b FLAG-SNAP-TERT immunopurified with a FLAG antibody from parental HEK 293 cells and various edited clones was revealed by western blot using antibodies against the FLAG-tag and TERT. Fluorescence was detected using SNAP Surface® 594 dye covalently bound to the SNAP-tag. All samples were supplemented with IP control (IP ctrl). c FLAG-SNAP-TERT immunopurified with a TERT-antibody from parental HEK 293 cells and various edited clones was revealed by western blot using antibodies against the FLAG-tag and TERT. A band with the size of endogenous TERT was also observed on the western blot of the modified clones, which should not arise from the NHEJ allele as the start codon was removed by the indel. One possibility is that the original start codon for the endogenous TERT on the FLAG-SNAP allele was still used to some extent instead of the start codon before the FLAG-tag sequence. Fluorescence detection and IP ctrl is as in panel (b). d Western blots and fluorescence imaging, comparing the IP efficiency of FLAG-SNAP-TERT using the anti-FLAG (F) or anti-TERT (T) antibody. e Direct telomerase activity assay of telomerase purified from parental HEK 293 cells and various edited HEK 293 clones with the anti-FLAG or anti-TERT antibody. Note lack of telomerase activity in lane 1 because no FLAG-labeled TERT is expressed in the parental cells. LC1 and LC2 are two oligonucleotide loading controls. f Telomeric restriction fragment length analysis by Southern blot of parental HEK 293 cells and several edited clones expressing FLAG-SNAP-TERT. Cells were harvested at the indicated time points. Average telomere lengths are plotted in the lower panel. Clones 3 and 4, which have higher levels of telomerase activity as shown in panel (e), have elongated telomeres. g Direct telomerase activity assay of telomerase purified from parental HEK 293 cells and various edited HEK 293 and HeLa clones using the anti-TERT antibody. Cells were transfected with a telomerase RNA expression plasmid (TR OE) to assure that FLAG-SNAP-TERT was the limiting component for telomerase assembly. IP ctrl was included in one sample after cell lysis to confirm that it did not affect telomerase activity. LC1 and LC2 are two oligonucleotide loading controls. Quantitative analysis of the data is plotted in the lower panel