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. 2007 Jul;13(7):1132–1139. doi: 10.1261/rna.474707

FIGURE 1.

FIGURE 1.

(A) Time course of TbAGO1 mRNA disappearance after removal of tet. TbAGO1KOc cells were washed twice in medium and then put back in culture in tet-minus medium for the times indicated above each lane. RNA was extracted and analyzed by Northern blot using a TbAGO1 coding-region probe. Note that two hybridizing bands are seen in lane 1; they are derived from utilization of two alternate 3′-splice sites present upstream of the TbAGO1 coding region. Control: the membrane was stripped and rehybridized with a probe detecting the mitochondrial large ribosomal RNA. (B) Decay of TbAGO1 mRNA in wild-type trypanosomes. Wild-type trypanosomes were incubated with the transcription inhibitor actinomycin D (AMD) as previously described (Shi et al. 2004b). RNA samples were prepared at the times indicated above each lane and analyzed by Northern blotting with an AGO1 coding-region probe. Control: hybridization to the 18S rRNA as a loading control. (C) Time course of the disappearance of TbAGO1 after tet removal. TbAGO1KOc cells were washed and incubated in tet-free medium as described in the legend of A. Whole-cell extracts were fractionated on a 6% SDS-PAGE and processed for Western blotting with anti-AGO1 antibodies (Shi et al. 2004b). Control indicates a cross-reactive protein. (D) Endogenous TbAGO1 is a stable protein. A cell line expressing BB2-TAP-tagged TbAGO1 (Shi et al. 2004b) was metabolically labeled with 35S-methionine as described in Materials and Methods. After a 2-h pulse, the label was chased with an excess of unlabeled methionine, and cytoplasmic extracts were prepared at the times indicated above each lane. TbAGO1 was then immunoprecipitated with anti-BB2 antibodies, the immunoprecipitates were separated by SDS-PAGE, and the radioactive bands were detected by autoradiography. The panel chase control shows a group of bands from the total protein samples run in parallel to show that the chase was effective. As a control for the efficacy of the immunoprecipitation, the membrane shown in the upper panel was analyzed by Western blotting with the anti-BB2 antibody, which detects the total amount of AGO1 protein present in the immunoprecipitates.