FIGURE 4.
(A) Inhibition of TbAGO1KOc RNAi competency after 4 h of growth in the absence of tet. Cells were washed free of tet and incubated for 2 h without or with addition of tet. Next, cells were simultaneously transfected with different amounts of α-tubulin and PFR dsRNA as indicated above each lane, and total RNA was isolated 2 h later. Ten micrograms of RNA was analyzed by Northern blotting with the following probes: a portion of the α-tubulin coding region (upper panel); a portion of the PFR coding region (middle panel); a portion of the actin mRNA as a loading control. The percent mRNA degradation was calculated as described in the legend to Figure 2. (B) The steady-state amount of TbAGO1 does not change within 4 h from tet removal. Cells were washed free of tet and immediately lysed with SDS-PAGE (time 0) or grown for 2 h, transfected with dsRNA, and then lysed 2 h later (time 4 h). The presence of AGO1 was revealed by Western blotting with the anti-AGO1 antiserum. (Con) A cross-reacting band used as a loading control. (C) The steady-state level of Ingi transcripts does not change during 5 h of incubation in the absence of tet. Ten micrograms of total RNA extracted from cells immediately after tet removal (time 0) or after 2 or 5 h of incubation in the absence of tet was analyzed by Northern blotting with an Ingi coding-region probe. (Con) 18S ribosomal RNA as a loading control.