Fig. 1.

TbTOR4 associates with both TbLST8 and Armtor. (A) Gel filtration elution profile of TbTOR4. Whole-cell lysates prepared in lysis buffer containing 0.3% CHAPS from wild-type bloodstream cells were loaded onto a Superose 6 sizing column. One-milliliter fractions were collected and processed for TbTOR4 immunoblotting. (B). Detection of TbLST8-interacting proteins in affinity purifications of TAP-LST8. Copurified material was resolved by SDS PAGE and visualized using Coomassie Brilliant Blue staining. An untagged cell line was used as negative control. (C) Endogenous TbTOR4 interacts with TbArmtor. Co-IP experiments were carried out using anti-TbTOR4 and anti-TbArmtor antibodies and conditions previously described (5). Western blotting of coimmunoprecipitated material revealed the binding of TbArmtor to TbTOR4. (D) TbTOR4 depletion halts cell proliferation. Growth curves of TbTOR4-depleted bloodstream trypanosomes after RNAi induction with doxycycline (Ind.) is compared with either uninduced (Unind.) or parental cell lines (Parent.) as controls. Cultures were diluted daily to 2.5 × 10⁴ cells/mL to maintain cell density within a range that supports exponential growth. (E) Western blot analysis of TbTOR4 expression in total cell extracts (5 ×10⁶ cells per lane) upon RNAi induction. Tubulin was used as a loading control.