Figure 6.

TbTRF and TelAP1 Co-IPs in procyclic cells. Co-IPs were performed in four biological replicates and enriched proteins were analyzed by MS. (A) Volcano plot representing TbTRF interactions in PCF cells. Six proteins were significantly enriched with TbTRF-Ty1 including four proteins, which were also found in the BSF TbTRF-Ty1 Co-IP: TbTIF2, Tb927.11.5550, Tb927.9.4000/3930, Tb927.6.4330. The whole dataset is summarized in Supplementary Table S4. (B) Volcano plot showing TelAP1 interacting proteins in PCF cells. Besides TelAP1 the telomere-associated candidate Tb927.6.4330 was enriched. (C) Western blot analysis of TbTRF-Ty1 Co-IP with anti-TelAP1 and anti-TbTRF antibodies. TbTRF was proteolytically degraded during the IP experiment as additional shorter bands appeared, which were detected by the monoclonal TbTRF antibody. Nevertheless, Ty1 epitope-tagged TbTRF was precipitated. TbTRF-Ty1 migrates slower in the SDS-PAGE than the WT TbTRF. TelAP1 could not be detected in the eluate. Twenty-fold more of the IP sample was loaded compared to IN and SN samples. (D) Western blot analysis of TelAP1 Co-IP with anti-TbTRF antibody confirmed interaction of TelAP1 with TbTRF in PCF stage. Again, TbTRF showed signs of proteolytic degradation after cell lysis. A smaller TbTRF fragment was co-purified with TelAP1. BSF and PCF whole cell lysates served as control and showed only one TbTRF product. Twenty-fold more of the pellet and IP sample were loaded compared to IN and SN samples. About 5% of TbTRF input was co-purified with TelAP1. S (starting material after lysis), IN (input), P (pellet), SN (supernatant), IP (immunoprecipitate).