The human EST1A TPR is sufficient for interaction with hTERT N- and C-termini. (A) hEST1A(114-824) but not hEST1A (114-631) interacts with hTERT N-terminus independently of hTR. Proteins synthesized in RRL were precipitated onto anti-FLAG resin (mock reactions contained no cDNA), washed, and treated with micrococcal nuclease (MNase). The precipitate was subjected to western blotting using anti-HA (top) followed by anti-FLAG (bottom) antibodies. Input, 20% of input HA2-hEST1A. Molecular mass (kDa) is indicated. One of 4 independent experiments is shown. MNase activity was verified via electrophoresis of DNA (data not shown). (B) Quantification of data in Panel A. The levels of hEST1A(114-824) or hEST1A(114-631) associated with hTERT fragments (or mock; no plasmid) are expressed as the normalized hEST1A signal relative to the normalized hTERT signal (see Methods; average ± S.D., n = 4). P-values represent statistically significant enrichment above the mock control. (C) Upper: Putative disordered loop in hEST1A TPR is dispensable for the hTERT interaction. Experiment as in Panel A, except that MNase was omitted. FLAG-Akt was used as a non-specific control. Protein complexes were precipitated onto anti-FLAG or anti-c-MYC agarose. Input, 25% of input HA2-hEST1A. Blots were probed with anti-HA antibody, stripped, and reprobed with anti-FLAG antibody. Lower: as above, performed with hEST1A (a.a. 114-749), a fragment that exhibited non-specific interactions (lanes 16, 18-20). (D) Mutation of two DAT regions of hTERT does not affect interaction with hESTA(114-824). Experiment performed as in Panel C. Amino acid substitutions (NAAIRS) starting at amino acid 92 or 122, as indicated.