Evo 6 scientific grade ccd camera

The interaction between PAR-2 and GSP-1/-2 was assessed using a GAL4-based system (Gateway, Invitrogen) using the MAV203 yeast strain. Full-length cDNAs of GSP-1 and GSP-2 were fused to the GAL4 DNA binding domain (Bait plasmid). A PAR-2 (1-335) fragment, both wild-type and mutant (RAFA), was fused to the GAL4 activation domain (Prey plasmid). The PAR-2 wild-type and mutant fragments and the GSP-1 and GSP-2 full length were first cloned into the pDONR201 and subsequently transferred to the pDEST22 vector (GAL4AD) and pDEST32 (GAL4DBD), respectively, using Gateway technology. Mutations were inserted by Pfu site-directed mutagenesis. A list of plasmids and primers used for the Y2H is provided in Tables S6 and S7, respectively. Transformants were selected on synthetic-defined medium (lacking leucine and tryptophan) plates. The interactions were tested by spotting single colonies containing the desired plasmids on a medium lacking leucine, tryptophan, and histidine and containing 50 mM of 3AT (3-amino-1,2,3-triazole; Sigma-Aldrich). Pictures of the plates were taken using the Fusion FX6 EDGE Imaging System (Vilber) equipped with an Evo-6 Scientific Grade CCD camera.