Yeast two-hybrid (Y2H) assay
To test protein-protein interactions coding sequences of CPT3 and LEW1
were subcloned into the pENTR/D-TOPO vector and next recombined into Y2H
vectors (pGADT7-GW and pGBKT7-GW) using LR Clonase II. Selected
constructs were transformed into S. cerevisiae AH109 strain [MATa, trp
1- 901, leu2 - 3, 112, ura 3 - 52, his3 - 200, gal4△, gal80△, LYS2:
GAL1UAS - GAL1TATA - HIS3, GAL2UAS - GAL2TATA - ADE2, URA3: MEL1UAS -
MEL1TATA – LacZ] using the lithium acetate method. Double
transformant colonies selected by colony PCR were grown in media lacking
leucine and tryptophan (-Leu/-Trp). Serial dilutions of the selected
double transformants were grown in plates lacking leucine, tryptophan
and histidine (- Leu/-Trp/-His) supplemented with 1mM 3-AT
(3-Amino-1,2,4-triazole). The experiments were performed in at least
three replicates.
Y2H vectors pGADT7-GW (Addgene plasmid #61702;
http://n2t.net/addgene:61702; RRID:Addgene_61702) and pGBKT7-GW
(Addgene plasmid #61703; http://n2t.net/addgene:61703 ;
RRID:Addgene_61703) were a gift from Yuhai Cui (Lu et al., 2010).