Yeast Two-Hybrid Protein Interaction Screening

Yeast two-hybrid experiments were performed using the Matchmaker^TM GAL4-based two-hybrid system (Clontech⁶). cDNA sequences encoding RRM protein (full-length and fragments), TERT fragments, G2p, MT2A, HSP70-1, and OZF2 were subcloned from their entry clones into the destination vectors pGADT7-DEST and pGBKT7-DEST. Each bait/prey combination was co-transformed into Saccharomyces cerevisiae PJ69-4a and yeast two hybrid analysis was performed as described in Schrumpfová et al. (2014) (link). Protein expression was verified by immunoblotting using mouse anti-HA (kindly provided by Dr. Vojtěšek) or mouse anti-myc primary antibodies and HRP-conjugated anti-mouse secondary antibody (both Sigma-Aldrich⁷).