Yeast two-hybrid system
The Matchmaker Two-Hybrid System 2 was used (Clontech). All experiments were performed in the yeast reporter strain CG-1945. Bait-constructs were made using the vector pGBT9 (Clontech). The prey-constructs pACT2-AF6, pACT2-Erbin, pACT2-PICK1, and pACT2-PSD95 were kindly provided by Jean-Paul Borg (INSERM, Marseille, France), and were described in Audebert and Navarro et al., (AF6, Erbin, PICK1) [46] and in Saito et al., (PSD-95) [31]. The prey-constructs pACT2-Syntenin and pACT2-CASK were kindly provided by Pascale Zimmermann (University of Leuven & VIB, Belgium). An oligo(dT)- and randomly primed prey-cDNA library constructed with mRNA from 12.5 day embryonic mice using pACT2 as vector [50] was kindly provided by Kristin Verschueren and Danny Huylebroeck (University of Leuven & VIB, Belgium).
The prey-library was screened as follows: yeast strain CG-1945, containing a HIS3 and a lacZ reporter gene under the control of promoters containing GAL4-binding sites, was transformed with 66 μg of bait-DNA and 33 μg of prey-library-DNA using a LiAc high efficiency transformation protocol [51]. Transformants were grown for 10 days at 30°C on triple selective (lacking Trp, Leu and His) synthetic dropout (SD---) agar plates containing 5 mM 3-AT (Sigma).
Transformed His+ yeast colonies were restreaked on new SD--- agar plates and grown for another 1 to 2 days. Colony-lift filter assays were performed for the qualitative measurement of β-galactosidase activity according to standard protocols.