FIGURE 4.

Ubiquitination-deficient TPP1 mutants localize to telomeres. A, Western blot analysis of cell lines stably expressing TPP1(WT)-3xHA or ubiquitination-deficient mutants. Relative, normalized levels of TPP1–3xHA constructs and USP7 are indicated below the respective blots, indicating similar expression levels for the TPP1–3xHA constructs and efficient depletion of USP7 by shRNA. Average values and standard deviations are indicated. B, immunostaining of cells from A for TPP1–3xHA constructs and TRF1. TPP1(WT)-3xHA and ubiquitination-deficient mutants co-localize with TRF1. USP7 depletion does not change telomere association of TPP1(WT)-3xHA as observed in several independent experiments. C, chromatin immunoprecipitation of TPP1–3xHA constructs from cells in A. D, quantification of C. TPP1(WT)-3xHA and ubiquitination-deficient mutants precipitate telomeric DNA. Average values and standard deviations (error bars) are indicated. USP7 depletion does not change telomere association of TPP1(WT)-3xHA. Only one ChIP quantification was performed for USP7 depletion because the immunostaining in B consistently showed unchanged TPP1(WT)-3xHA telomere association in the absence of USP7. EV, empty vector; IF, immunofluorescence.