Figure 1.
Screen workflow for the identification of adenylyl cyclase type 5 (AC5) interaction network partners using unique bimolecular fluorescence complementation (BiFC) tagged cDNA library. A unique human brain cDNA library was cloned into the BiFC plasmid, such that each cDNA would be expressed linked to the C-terminal fragment of Venus (i.e., cDNA-VC). The BiFC tagged cDNA library was transiently expressed into CAD VN-AC5/D2L, and the development of heterologous sensitization initiated. Fluorescence-activated cell sorting was used to identify and isolate cells that exhibited an AC5-protein network interaction, as determined by a BiFC fluorescent signal. Next-generation sequencing (NGS) was used to identify the potential interacting proteins from the complementing cDNAs, followed by the siRNA knockdown of identified genes to confirm their role in heterologous sensitization.