Pdest32

Yeast two-hybrid screens were performed using the Gateway ProQuest Two-Hybrid System (Invitrogen) essentially as described by the manufacturer. To generate bait plasmids, AMPKα1 (encoding amino acids 10–559) and AMPKα2 (full length) in pENTR221 (Invitrogen) were transferred to the pDEST32 (Invitrogen) and transformed into MAV203 Saccharomyces cerevisiae strain. The ProQuest pre-made human fetal brain cDNA library and a human liver cDNA library in EXP-AD502 (Invitrogen) were transformed into pDEST32-AMPKα1 or pDEST32-AMPKα2 yeast strains, to perform screenings. AMPKα1/brain (4.2 × 10⁶), AMPKα2/brain (2.9 × 10⁶), AMPKα1/liver (2.1 × 10⁶) and AMPKα2/liver (2.0 × 10⁶) colonies were screened and positive clones were subjected to sequencing.

The ORF of PtrICE1 generated by RT-PCR using primer pair GSP3 (Supplementary Table S1) was digested with BamHI and XhoI and inserted into pENTR3C (Invitrogen). The recombinant vector (pENTR3C-PtrICE1) was fused in frame downstream of the yeast GAL4 DNA-binding domain (BD) of vector pDEST32 (Invitrogen). The fusion vector and negative control (pDEST32) were transformed independently into yeast MaV203 strain (Invitrogen) according to the manufacturer’s instructions. The transformed yeast cells were plated for 3 d on SD/–Leu/–Trp or SD/–Leu/–Trp/–His medium added with or without 5 and 15mM 3-aminotriazole (3-AT), followed by growth observation.
A yeast one-hybrid assay (Clontech) was performed to investigate whether PtrICE1 could bind to the MYCR sequence. The PtrICE1 ORF was amplified using the primer pair GSP4 (Supplementary Table S1) and fused to the GAL4 activation domain (AD) in the pGADT7 vector to create pGADT7-PtrICE1. A 66bp DNA fragment composed of triple tandem repeats of a sequence containing MYCR (CACATG) was inserted into the pHIS2 vector, generating a recombinant construct of pHIS2-MYCR. Thereafter, pGADT7-PtrICE1 and pHIS2-MYCR were co-transformed into yeast cells (strain Y187), which were grown for 3 d on SD/–Leu/–Trp/–His medium with or without 15mM 3-AT.

For the transactivation assay, the full-length ORF of PubHLH1 was amplified by PCR using primers (GSP5) containing either a BamHI or XhoI restriction site, and the amplicon was inserted into the same enzyme recognition sites of pENTR3C (Invitrogen). The PubHLH1 fragment was then fused downstream of the yeast GAL4 DNA-binding domain in pDEST32 by recombination reactions (Invitrogen). The fusion vector and the negative control (pDEST32) were independently expressed in yeast strain MaV203 (Invitrogen) according to the manufacturer’s instructions. The transformed yeast strains were placed on SD/–Leu/–Trp or SD/–Leu/–Trp/–His medium with or without different concentrations of 3-amino-1,2,4-triazole (3-AT, 0, 5, and 15 mM) and cultured for 3–4 days to test the expression of the reporter gene HIS3.