gal4 activating domain ad  (TaKaRa)

Journal: Plant Biotechnology Journal

Article Title: Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso) 4E in Beta vulgaris

doi: 10.1111/pbi.14333

Figure Lengend Snippet: GAL4 based YTH experiments for interaction between poleroviral VPg/VPg + C‐term and Bv‐eIF4E. Bv‐eIF4E was fused to the binding domain (BD) and VPg/VPg + C‐term to the activation domain (AD) for PPI evaluation. Positive control: AD‐p53 with BD‐T; Negative control: AD‐p53 with BD‐Lam. Two plasmids containing AD and BD fusion proteins, respectively, were co‐transformed into yeast. To test for autoactivation, yeast strain Y2HGold was co‐transformed with the plasmids encoding the proteins of interest and the AD or BD plasmids lacking a fusion protein. Single colonies were resuspended in water and diluted 1 × 10 0 –1 × 10 −3 . Then, 5 μL of each dilution was spotted on control medium SD (‐W, ‐L) and interaction media SD (‐W, ‐L, ‐H) and SD (‐W, ‐L, ‐H, ‐Ade). Spotted yeast was incubated at 30 °C for 3 days (control medium) or 5 days (selection medium), respectively. Only 1 × 10 −2 dilution of spotted yeast is displayed. Expression of the fusion proteins was confirmed by Western blot analysis (Figure ).

Article Snippet: The GAL4 based Matchmaker Gold YTH (Takara Bio Europe; Saint‐Germain‐en‐Laye) was used according to the manufacturer's instructions using the reporter genes HIS3 and ADE2 to detect PPI.

Techniques: Binding Assay, Activation Assay, Positive Control, Negative Control, Transformation Assay, Control, Incubation, Selection, Expressing, Western Blot

Journal: Plant Biotechnology Journal

Article Title: Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso) 4E in Beta vulgaris

doi: 10.1111/pbi.14333

Figure Lengend Snippet: GAL4 based YTH experiments tested for interaction between (a) BMYV‐VPg or (b) BChV‐VPg + C‐term and Bv‐eIF(iso)4E mutated in its mRNA cap‐binding amino acids. Mutations exchanging proteinogenic amino acid to leucine within Bv‐eIF(iso)4E were introduced by site directed mutagenesis and are indicated with their position in the protein sequence. The Bv‐eIF(iso)4E mutant was fused to the binding domain (BD) and VPg/VPg + C‐term to the activation domain (AD) for PPI evaluation. Positive control: AD‐p53 with BD‐T; Negative control: AD‐p53 with BD‐Lam. Yeast was co‐transformed with the two plasmids containing the AD and BD fusion proteins. To test for autoactivation, yeast cells were co‐transformed with the plasmids encoding the proteins of interest and the AD or BD plasmids lacking a fusion protein. Single colonies were resuspended in water and diluted 1 × 10 0 –1 × 10 −3 . 5 μL of each dilution was spotted on control medium SD (‐W, ‐L), interaction media SD (‐W, ‐L, ‐H) and SD (‐W, ‐L, ‐H, ‐Ade). Spotted yeast was incubated at 30 °C for 3 days (control medium) or 5 days (selection medium). WT = wildtype Bv‐eIF(iso)4E. Only 1 × 10 −2 dilution of spotted yeast is displayed. Expression of the fusion proteins was confirmed by Western blot analysis (Figure ).

Article Snippet: The GAL4 based Matchmaker Gold YTH (Takara Bio Europe; Saint‐Germain‐en‐Laye) was used according to the manufacturer's instructions using the reporter genes HIS3 and ADE2 to detect PPI.

Techniques: Binding Assay, Mutagenesis, Sequencing, Activation Assay, Positive Control, Negative Control, Transformation Assay, Control, Incubation, Selection, Expressing, Western Blot