(A) Coomassie-blue-stained SDS-PAGE gel of purified POT1/TPP1-N complex and hOB-hDBD fusion proteins used in (B).
(B) Direct telomerase activity assay. Lane 1: telomerase extract alone; lane 2 includes human POT1 and TPP1-N; and lanes 3–6 include the indicated hOB-hDBD variant.
(C) Quantitation of telomerase processivity from the indicated number of replicates (n), of which (B) is representative. Statistical significance was scored with a two-tailed Student’s t test. Error bars indicate SD. **p value between 0.001 to 0.01; ***p < 0.001.
(D) Pull-down of FLAG-TPP1 (WT or ΔNOB) and Myc-POT1 or Myc-TIN2 transiently expressed in HeLa cells on anti-FLAG conjugated beads. “Input” and “FLAG-IP” immunoblots refer to soluble lysates before incubation with anti-FLAG beads and anti-FLAG beads after overnight immunoprecipitation, respectively. POT1 was not detected in the input sample but was detected in FLAG-IP samples that contained TPP1, consistent with POT1 stabilization by TPP1. Asterisks indicate POT1- and TIN2-derived degradation products.