Pgad424

Enhanced green fluorescent protein (EGFP) cDNA from pEGFP-C1 (Takara bio Inc.) was cloned into mammalian gene expression vector pCS2+ [36 (link)]. pCS2+p27 and pCSMT-cyclin E were provided by M. Ohtsubo. The p27 cDNA fused to N-terminal of EGFP was also cloned into pCS2+ to construct pCS2+p27-EGFP.
Doxycycline (Dox)-inducible exogenous p27 expression system was constructed using pRevTet-On and pRevTRE (Takara bio Inc., Kusatsu, Japan.). Yeast two-hybrid system was constructed using pGBTK [37 (link)] and pGAD424 (Takara bio Inc., Kusatsu, Japan) as a bait and a prey protein expression vector, respectively. Stable overexpression and stable knockdown for NPM1 were carried out using pLPCX and pSIREN Retro-Q (Takara bio Inc., Kusatsu, Japan), respectively.

We used pGAD424 and pGBT9 plasmids (Takara Bio USA, Mountain View, CA, USA) for the construction of prey and bait vectors, respectively. As described above, the CDS of each gene was cloned and introduced into pGAD424 or pGBT9. Primers used for PCR are listed in Table S1.
Both prey and bait vectors were introduced into the yeast strain PJ69-4A and were cultured in liquid media lacking Leu and Trp (-LW). Yeast cells were grown and collected at a density of 0.4–0.6 OD/mL. Cells were then diluted to 6 × 10² cells/μL, and then 10-fold serial dilutions were prepared. Diluted cells (10 μL) were spotted on -LW and -LWH (i.e., media lacking Leu, Trp, and His). When necessary, an appropriate concentration of 3-amino-1,2,4-triazole (3-AT) was added to the plates. Cells were grown for 3 days at 30 °C.

PCR products encoding full-length TbPex3 and full-length HsPex3 were cloned in-frame and downstream of the DNA-binding domain (BD) of the GAL4 transcriptional activator in pGBT9 (Clontech). Full-length TbPex19 and full-length HsPex19 were cloned in-frame and downstream of the activation domain (AD) of the GAL4 transcriptional activator in pGAD424 (Clontech). The S. cerevisiae strain HF7c deleted for the PDR5 gene (HF7c pdr5Δ) encoding the major drug efflux pump of yeast (Golin and Ambudkar, 2015 (link)) was transformed with plasmids, and transformed cells were grown on synthetic dropout medium agar lacking leucine and tryptophan (-Leu -Trp) to determine total cell growth and on synthetic dropout medium agar lacking histidine, leucine, and tryptophan (-His -Leu -Trp) to determine growth of cells exhibiting protein–protein interaction between the AD-fusion and BD-fusion constructs.