Fig. 4.

TPP1 regulates TRF1 association with the TRF2/RAP1 complex in vivo. (a) Design of the pRetro-dual RNAi vector for TPP1 knockdown. TPP1 RNAi oligos were cloned between the head-to-head-positioned U6 and H1 promoters. (b) Reducing endogenous TPP1 levels by RNAi decreases TRF1 recruitment to the TRF2/RAP1 complex. (Left) Nuclear extracts (420 mM KCl extracted) were prepared from HT1080 cells that were transfected with pRetro-dual control and TPP1 RNAi vectors. The extracts were resolved by SDS/PAGE and Western-blotted with the indicated antibodies. The anti-Histone H1 antibody was used as a loading control (note that the siTPP1 #7 lane was slightly underloaded). (Right) Nuclear extracts from HT1080 cells expressing pRetro-dual siTPP1 #1 and siTPP1 #7 were immunoprecipitated with anti-RAP1 antibodies, and Western-blotted with anti-RAP1 and anti-TRF1 antibodies.