Pgbkt7 yeast vector

Yeast two-hybrid (Y₂H) screening was performed using the Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual (Clontech). First, the open reading frame (ORF) of Clg2p was amplified with primers 8F/R and inserted into the EcoRI and BamHI sites of the pGBKT7 yeast vector (Clontech) to create a bait. An AD fusion library in pGADT7-rescuing cDNA from C. lunata was constructed and transferred into the yeast strain AH109 as described in Jing et al.58 . The SD/-Ade/-His/-Leu/-Trp/X-α-Gal medium was used to screen and confirm expression. Then, plasmid DNA from the positive clones was isolated from yeast, transformed into E. coli and confirmed by sequencing.
Sequences from positive clones were analysed using BLASTx (http://www.ncbi.nlm.nih.gov). The predicted ORFs of positive colonies were extracted from the genome of C. lunata (http://www.ncbi.nlm.nih.gov/nuccore/JFHG00000000) and confirmed by RT-PCR with cDNA as a template. The percentage of similarity and identity of the known sequences were determined using the MatGAT programme. The conserved domains of the deduced proteins were analysed by CDD Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and Pfam (http://pfam.xfam.org/).

As bait, the N-terminal region of RIPK4 was cloned into the pGBKT7 yeast vector (Clontech, USA) in fusion with the Gal4 DNA binding domain, and the construct was sent to the German Cancer Research Center (DKFZ, Genomics and Proteomics Core Facilities - Protein Interaction Screening W150, Germany) for a large-scale Y2H screen. The human keratinocyte cDNA library, which was expressed in fusion with the Gal4 activation domain from vector pACT2.2 (Clontech, USA), was used as prey. Bait and prey proteins were expressed together in the AH109 yeast strain (Clontech, USA) and positive clones were identified by activation of HIS3 and MEL1 reporters that are controlled by GAL promoters. The Y2H screen was repeated three times with 0.4 mM, 0.2 mM, and 0.1 mM 3-aminotriazole (3AT) containing selective medium to determine strong and weak interactions. Library inserts were isolated by PCR and analyzed by DNA sequencing.