2.10 Yeast two-hybrid analysis and transcription activation activity
A yeast two-hybrid assay was performed using a HybriZAP-2.1 two-hybrid vector system (Stratagene, https://www.agilent.com/) as described previously (Matsuoka, Yasufuku, Furuya, & Manmori, 2015). The full-length coding regions of CRCT , GF14A and GF14Bwere amplified by RT-PCR using gene-specific primers (Table S1). The PCR product of CRCT was cloned into the pAD-GAL4-2.1 vector for the expression of GAL4 transcriptional activation domain fusion proteins. The PCR products of GF14A and GF14B were inserted into the pBD-GAL4 Cam vector for the expression of GAL4 DNA-binding domain fusion proteins. Each pair of bait and prey vectors was cotransformed into yeast (strain YRG-2). The transformation was confirmed by growth on the synthetic media plate lacking Leu and Trp (SD2-). The protein interaction was monitored by growth on the synthetic media plate lacking Leu, Trp and His (SD3-).
Transcriptional activation activity was analyzed using the two-hybrid vector system without pAD-GAL4-2.1 vector. Eight different CRCTfragments were amplified by PCR using primers listed in Table S1 and cloned into the pBD-GAL4 Cam vector. The vectors were transformed into the yeast. The transformation and transcription activation activity were monitored by growth of yeast on the synthetic media plate lacking Trp (SD1-) and Trp and His (SD2-), respectively.