Fig 6.

Expression of a T. cruzi CITFA-7-HA transgene did not rescue trypanosome proliferation from a T. brucei CITFA-7 knockdown. (A) Culture growth of BFs of noninduced (-Dox) and TbCITFA-7-silenced (+Dox) cells that express TcCITFA-7-HA. (B) Ethidium bromide staining of rRNA and semiquantitative RT-PCR analysis of listed mRNAs at 0, 24, and 72 h of doxycycline induction. Note that TbCITFA-7 mRNA was targeted by the knockdown (KD). (C) Immunoblot analysis of whole-cell lysates prepared from noninduced cells or cells that were induced for 24 h. TbCITFA-7 and TFIIB were detected with polyclonal antibodies against the endogenous proteins, and TcCITFA-7–HA was detected with a monoclonal anti-HA antibody. (D) Sedimentation of extract by ultracentrifugation in a 10 to 40% linear sucrose gradient. Fractions 4 to 20, taken from top to bottom, were analyzed by immunoblotting. For comparison, sedimentations of TEV protease (29 kDa), Taq DNA polymerase (95 kDa), IgG (150 kDa, 6.6S), the TRF4-SNAPc-TFIIA transcription factor complex (TST) (∼230 kDa) (30), apoferritin (AP) (444 kDa, 17S), and thyroglobin (TG) (660 kDa, 19S) were analyzed in parallel gradients (arrowheads).