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. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: Mol Biochem Parasitol. 2017 Sep 28;218:4–15. doi: 10.1016/j.molbiopara.2017.09.003

Fig. 2.

Fig. 2

Constitutive expression (CE) of the FL and N-terminal deletion mutants of TbTim17 in Tim17 knockdown (KD) T. brucei. (A) The schematic diagram of the pHD1344 constitutive expression vector for FL and N-terminal deletion mutants and the tetracycline-inducible p2T7-177/Tim17(3′UTR) RNAi construct. SAS, splice acceptor site; Puro R and Phleo R, puromycin and phleomycin resistance genes, respectively; Tet R, tetracyclin repressor. Arrows indicate the direction of transcription. (B) Growth kinetics of Tim17KD, Tim17KD/Δ20-Tim17CE, Tim17KD/Δ30-Tim17CE, and Tim17KD/FL-Tim17CE in the absence (uninduced) and presence (induced) of doxycycline. Cell numbers were counted up to 10 days and the log of cumulative cell numbers was plotted against time. Results represent three independent experiments. (C) Immunoblot analysis of total cellular proteins harvested at different time points (0–4 days) after induction with doxycycline. TbTim17KD cells, and the double-transfected T. brucei cells Tim17KD/Δ20-Tim17CE, Tim17KD/Δ30-Tim17CE, and Tim17KD/FL-Tim17CE were grown in the presence of doxycycline for induction of Tim17 RNAi targeted to the 3′UTR of the TbTim17 transcript. Proteins were analyzed by SDS-PAGE and immunoblot analysis using antibodies for T. brucei Tim17, VDAC and also for the myc epitope tag. Equal numbers of cell (5 × 106/ml) were loaded per lane. (D) The intensity of protein bands for the endogenous (Endo) Tim17 (recognized by anti-Tim17 antibody) and the ectopically (Ecto) expressed FL and N-terminal deletion mutants (recognized by anti-myc antibody) were quantitated by densitometry, normalized with the levels of VDAC in the corresponding samples and were presented as percent of the endogenous TbTim17 levels at 0 time point in the corresponding cell line. Standard errors were calculated from 3 independent experiments. (E) The Tim17KD/FL-Tim17CE, Tim17KD/Δ20-Tim17CE, and Tim17KD/Δ30-Tim17CE cells were grown in the presence of doxycycline for 96 h. Cells were harvested and sub-cellular fractionation were performed. Total (lane T), Cytosolic (lane C), and mitochondrial (lane M) fractions were analyzed by SDS-PAGE and immunoblotting using anti-myc, -Tim17, -VDAC, and -PP5 antibodies.