Yeast Two-Hybrid Assay and Co-IP Protocol

Yeast transformation was performed using S. cerevisiae Direct Transformation Kit (Wako, Japan). Yeast two-hybrid assays were performed using MATCHMAKER Two-Hybrid System3 (Clontech) as previously described (Kayano et al., 2018 (link)). Briefly, yeast strain AH109 was transformed with prey and bait vectors, pGADT7 and pGBKT7 (Supplementary Table S2) and transformants were isolated on SD medium lacking leucine and tryptophan. Transformants were then cultured on SD medium lacking leucine, tryptophan, histidine and adenine (-LTHA), and growth on the latter indicates an interaction between bait and prey. Co-immunoprecipitation assay was performed as described by Xu et al. (2015) (link) using monoclonal antibodies against GFP (mFX75 012-22541, Wako) and FLAG (anti-DYKDDDDK 018-22381, Wako).