FIGURE 6.

Ubiquitinated TPP1 is able to interact with TIN2, POT1, CTC1-STN1, and telomerase. A, binding assay using TPP1(WT)- or TPP1(10R)-3xHA-His and TIN2 purified from HEK293T cells as described under “Experimental Procedures” and analyzed by Western blotting. Vertical lines separate different parts of the same gel. Both TPP1 constructs are able to interact with TIN2–3xFLAG upon denaturation/refolding during the purification process. Purified ubiquitinated (ubiquit.) TPP1–3xHA or in vitro deubiquitinated (deub.) TPP1–3xHA was incubated in binding assays with TIN2–3xFLAG (B), POT1–3xFLAG (C), telomerase (D), or CTC1-V5/STN1–3xFLAG (E) purified from HEK293T cells as described under “Experimental Procedures.” Vertical lines separate different parts of the same gel. Asterisks indicate USP7–3xFLAG used for deubiquitination. Western blot analysis reveals no difference in binding of ubiquitinated or deubiquitinated TPP1–3xHA to the factors tested.