Matchmaker 3 yeast two hybrid system

Human cDNA fragments encoding for EB1 (NM_012325), EB3 (NM_012326), SKAP (NM_033286.2), Astrin (NM006461.3) were subcloned into pGBT9 and pGAD424 (Clontech). SKAP point mutants (¹¹³LP to NN and RAT¹⁰⁸ into either RAA or RAE) were created by PCR mutagenesis and confirmed by DNA sequencing. Yeast two-hybrid protocols were based on the Matchmaker 3 yeast two-hybrid system (Clontech).

Protein-protein interaction was tested as previously described using the Matchmaker 3 yeast two-hybrid system (Clontech). The growth of yeast colonies containing both prey and bait vectors is shown as a control on Figure 2 on selective dropout medium lacking leucine and tryptophan (SD-LW) and interactions were selected on selective dropout medium lacking leucine, tryptophan and histidine (SD-LWH). Each combination was tested in triplicate. The TEV HAT VPg was fused to the binding domain (BD) of the GAL4 while the different eIF4E1 and eIFiso4E were fused to the activation domain (AD). All plasmids have been described previously [17 (link),20 (link)].