The TrxG protein FSH interacts biochemically with ASH1. (A) Map of inducible, tandem-tagged ASH1 expression construct used to establish stable cell line. Amp: ampicillin resistance, bla: beta-lactamase promoter; FH: 3xFLAG-8xHIS tandem affinity tag; Hyg: hygromycin resistance; MT: metallothionein promoter;, ori: origin of replication; P copia: copia promoter. (B) Outline of ASH1 purification scheme. ESI-MS/MS: electrospray ionization tandem mass spectrometry; FLAG-AC: FLAG affinity chromatography; IEC: ion exchange chromatography; IMAC: immobilized metal affinity chromatography. (C) FH-ASH1 dependent enrichment of proteins by purification scheme. Purified FH-ASH1 sample was size separated and silver stained together with control sample. (D) Summary of mass spectrometry results; only the top two enriched proteins are shown (for complete results see Additional file 1). (E) Maps of protein fragments used for coimmunoprecipitation experiments. (F) Coimmunoprecipitation of FSH-S through ASH-A fragment. S2-DRSC cells were cotransfected with the indicated constructs, and whole cell lysates and precipitated proteins were immunoblotted using the indicated antibodies.
Kockmann et al. Genome Biology 2013 14:R18 doi:10.1186/gb-2013-14-2-r18