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. 2018 Apr 25;131(8):jcs211730. doi: 10.1242/jcs.211730

Fig. 6.

Fig. 6.

MiRF172 and TAC102 after p197 RNAi depletion and recovery after removal of tet in γL262P p197 RNAi BSF T. brucei cells. (A) Colocalization of MiRF172–PTP with TAC102 in γL262P p197 RNAi BSF cells. Localization of MiRF172–PTP (magenta) and TAC102 (green) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of γL262P p197 RNAi BSF T. brucei cells. MiRF172–PTP was detected with anti-Protein A antibody. TAC102 was detected with anti-TAC102 monoclonal mouse antibody. The kDNA and the nucleus were stained with DAPI (cyan). The inset shows a higher magnification view. (B) TAC recovery experiment in γL262P p197 RNAi BSF T. brucei cells. To detect MiRF172–PTP, TAC102 and DNA the same antibodies and reagents as in A were used. The pictures were obtained under the same conditions as in A. The basal bodies (red) were detected with the YL1/2 monoclonal antibody. - tet, uninduced cells; d5 post induction, MiRF172-depleted cells at day 5 of RNAi (RNAi was induced by addition of tet); d4 post recovery, after 5 days of RNAi, tet was removed and cells were grown for 4 additional days. (C) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P p197 RNAi induced and uninduced cells (n≥113 for each time point). K, kDNA; N, nucleus. (D) Quantitative analysis of TAC102 in γL262P p197 RNAi cells without tet (no tet), with tet at day five (d5 p.i.) as well as 2 days after removal of tet (post recovery; d2 p.r.) and at day 4 post recovery (d4 p.r.) (n≥105 for each time point). (E) Quantitative analysis of the MiRF172–PTP signal in in γL262P p197 RNAi cells as in D (n≥105 for each time point). (F) Western blot analysis of γL262P p197 RNAi BSF cells. Total protein isolated from uninduced cells (−tet), cells induced with tet for 5 days (d5 p.i.) and cells released from p197 RNAi at day 2 (d2 p.r.) and day 4 post recovery (d4 p.r.) was used. C-terminally PTP-tagged MiRF172 was detected with the anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. EF1α serves as a loading control. Arrowheads point to the TAC102 and MiRF172 signals. PH, phase contrast. Scale bars: 5 µm.