Tethering assays were done using cells expressing mRNAs with a chloramphenicol acetyltransferase (CAT) open reading frame and a truncated version of the trypanosome actinA 3'-UTR, with five boxB sequences immediately upstream of the 3'-UTR (Mugo and Erben, 2020). Lines with tetracycline-inducible expression of different versions of CFB2 (as shown in Figure 7) were made, and CAT was assayed with or without tetracycline addition. CAT activity was performed as previously described using 14C-labelled butyryl coenzyme A as the labelled substrate (Mugo and Erben, 2020). Yeast two-hybrid assays were done using the Matchmaker Yeast Two-Hybrid System (Clontech) following the manufacturer's recommendations. The DNAs of the protein ORFs were PCR-amplified and cloned into pGBKT7 or pGADT7. Subsequent mutations of the ORFs were achieved via site-directed mutagenesis (Q5 Site-Directed Mutagenesis, New England Biolabs). The plasmids were co-transformed pairwise into AH109 yeast strains, and co-transformants were selected on double drop-out medium (minimal SD media lacking Trp and Leu). Positive interactions were indicated by growth on quadruple drop-out medium (minimal SD media lacking Trp, Leu, His, and Ade). The interaction between murine p53 and SV40 large T-antigen served as the positive control, with LaminC and the SV40 large T-antigen as the negative bait (DNA-binding domain) and prey controls, respectively. Expression of bait and prey proteins was checked by detection of HA and c-myc epitopes, respectively.

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