Pgbkt7 bait vector

A Matchmaker gold two-hybrid system (Clontech) was used to screen host proteins that interact with the AnkH protein per the instructions of the manufacturer. Full-length AnkH coding sequence was amplified, subjected to sequencing, cloned into the pGBKT7 bait vector (Clontech), and transformed into yeast strain AH109 (Clontech). A normalized universal human cDNA library in pGADT7 was purchased (Clontech) for use as potential prey targets. The library and bait containing AH109 were mated, and the resulting colonies were screened per the instructions of the manufacturer. Plasmids from positive clones were isolated using yeast lysis buffer and glass beads. Isolated prey plasmid and bait plasmid were used to cotransform the AH109 yeast strain. Transformants were selected by growing the yeast on SD medium lacking His, Leu, and Trp (SD-His/-Leu/-Trp) (Clontech). Colonies that tested positive were then transferred to SD-Ade/-His/-Leu/-Trp plates containing 5-bromo-4-chloro-3-indoxyl-α-d-galactopyranoside (X-α-Gal) (GoldBio). Blue colonies were selected for plasmid isolation. Isolated plasmids were then sequenced to identify the human genes.

AgMDL1 was amplified from Sua5B cDNA and cloned into the pGBKT7 bait vector using the primers described in S1 Table according to the Matchmaker Gold Yeast Two Hybrid System user protocol (Clontech). For cDNA library construction, total RNA was isolated from Sua5B cells, reverse-transcribed into cDNA, and cloned into the pGADT7 prey vector using the Make Your Own Mate and Plate Library System (Clontech). pGBKT7 (bait) and pGADT7 (prey) vectors were transformed in the Y2HGold and Y187 yeast strains, respectively. Screening was performed according to the Matchmaker Gold Yeast Two-Hybrid System user protocol (Clontech) using either double dropout synthetic medium (leucine and tryptophan) or quadruple dropout synthetic medium (leucine, tryptophan, adenine, histidine) supplemented with aureobasidin and X-α-Gal (Clontech). Positive clones were sequenced and blasted against the An. gambiae genome in Vectorbase. Interacting proteins were confirmed using standard pulldown assays.

Yeast Two-hybrid experiment was performed as described previously (Yang et al., 2015 (link)). Briefly, cDNA encoding interesting genes were amplified from w¹¹¹⁸ ovary cDNA and cloned into either pGBKT7 bait vector or pGAD prey vector (Clontech). The pGBKT7 and pGAD plasmid carrying interesting genes were co-transformed into AH109 yeast cells according to a standard procedure. Colonies appearing on media lacking tryptophan and leucine (SC-WL) were picked onto selection plate lacking tryptophan, leucine and histidine (SC-WLH) or tryptophan, leucine, histidine and adenine (SC-WLHA) to determine proteins interaction.