Two-Hybrid Screening of EFA-6 Interactors

We performed two-hybrid screening as described (Wang et al., 2013 (link)). We cloned EFA-6 full-length cDNA and fragments into a pMB27-Gal4-BD-gtwy vector, derived from the pPC97-Gal4-BD vector. We transformed baits into yeast strain Y8930, and mated these to a pPC86-Gal4-AD prey library of mixed-stage C. elegans cDNAs in strain Y8800. Plasmids for two-hybrid experiments are listed in Supplementary file 1B. We screened >2 × 10⁶ independent colonies per bait, and identified interacting cDNAs by plasmid amplification and sequencing. To test specific interactions we cloned the appropriate full length or fragment cDNAs into the pACT2 (Gal4 activation domain) or pBTM116 (LexA DNA-binding domain) vectors (Clontech, Mountain View, CA) and co-transformed constructs into yeast strain L40. We grew transformed yeasts on agar plates with SD medium (synthetic minimal medium) lacking leucine and tryptophan; interactions were examined on plates with SD medium lacking leucine, tryptophan, and histidine, with or without 3-AT.