Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from transfected construct in HEK293T cells.

synr was cloned into a vector that includes tRNA synthetase and tRNA pair to incorporate pBPA into aa 124Y (of the BH3 domain), or 141F (near the BH3 domain) to stabilize the interaction. It was then incubated with transfected HA-tagged Debcl and uv-crosslinked. Additionally, wild-type synr can pull down Debcl and Buffy simultaneously.

Sequential coIP. synr interacted with Debcl only after an initial immunoprecipitation with Buffy.

Source was cell extract of HEK293T cell line; bait produced from transfected construct; prey produced from transfected construct.