Telomerase enzymatic activities as determined in VA13+hTERC cells for naturally occurring hTERT variants.

(A) Representative TRAP gels showing the relative telomerase enzymatic activities obtained from the substitution mutations and the wild-type hTERT constructs either individually transfected (lanes 1-21, 45-50) or cotransfected (lanes 22-42, 51-56). Serial 5-fold dilutions of the transfected cell lysates (indicated by triangles) were assayed for each sample to ensure linearity of the PCR-based TRAP assay. Lane 43 shows a negative control composed of wild-type cell lysate denatured at 95°C for 5 minutes prior to assaying. Lane 44 shows PCR products amplified from the non-hTERC control TSR8 DNA template supplied in the TRAP kit. IC indicates PCR products amplified from an unrelated DNA template, which is included as an internal control for PCR amplification efficiency in each reaction. (B) Western blot of wild-type and various natural hTERT variants isolated from transfected cell lysates and probed with an anti-HA probe against the N-terminal HA tag at each of the hTERT constructs.