Lack of physical interaction between SBDS and telomerase complex components. (A) FLAG-tagged TERT construct was transfected into HeLa and HEK 293T cells. Left panel illustrates HeLa cell lysates transfected (+) or not transfected (−) with FLAG-TERT probed for FLAG-TERT with an anti-FLAG monoclonal antibody, indicating effective TERT expression. Immunoprecipitation with anti-FLAG antibody was performed and precipitates probed for SBDS (right panel). (B) TERT-SBDS interaction was further explored using the 2 hybrid system. Luciferase activity indicates close proximity of NF-κB and yeast GAL4. (C) Association between SBDS and TERC RNA. TERC RNA was synthesized in vitro and incubated with rabbit reticulocyte lysate (RRL) expressing FLAG-TERT, FLAG-SBDS, FLAG-SBDS-84X, or empty vector (left panel; lane 1, RRL expressing FLAG-SBDS-84X; lane 2, RRL expressing FLAG-SBDS; and lane 3, RRL expressing FLAG-TERT). Following FLAG immunoprecipitation, RNA was extracted from the precipitates and examined for TERC. Right panel illustrates Northern blot for TERC: lane 1, non-transfected RRL supernatant; lane 2, FLAG-SBDS-84X-transfected RRL supernatant; lane 3, FLAG-SBDS-transfected RRL supernatant; lane 4, FLAG-TERT-transfected RRL supernatant; lane 5, non-transfected RRL precipitate; lane 6, FLAG-SBDS-84X-transfected RRL precipitate; lane 7, FLAG-SBDS-transfected RRL precipitate; lane 8, FLAG-TERT-transfected RRL precipitate. Despite the low expression FLAG-TERT by the RRL, it efficiently precipitated TERC. In contrast, large amounts of FLAG-SBDS and FLAG-SBDS-84X failed to precipitate TERC to any appreciable degree.