gal4 activating domain ad  (TaKaRa)

Journal: bioRxiv

Article Title: SPOROCYTELESS/NOZZLE acts together with MADS-domain transcription factors to regulate an auxin-dependent network controlling the Megaspore Mother Cell development

doi: 10.1101/2025.03.11.641985

Figure Lengend Snippet: ( A ) Yeast-two-hybrid assay showing interaction between SPL/NZZ and MADS-domain TFs (SEP3, STK, AG). The controls are shown in the . Interactions are tested on media depleted of histidine (-W -L -H), or histidine and adenine (-W -L -H -A). For each interaction on the different media, yeast has been spotted at four different concentrations according to a serial dilution (OD 0,5; 1:10; 1:100; 1:1000). ( B, C ) Yeast-three-hybrid assay showing the formation of SPL/NZZ, STK and SEP3 complex, by fusing SPL/NZZ either to the GAL4 BD ( B ) or to the TFT ( C ). Interactions are tested on a medium depleted of histidine (-W -L -H -A). For each interaction, yeast has been spotted at four different concentrations according to a serial dilution (OD 0,5; 1:10; 1:100; 1:1000). ( D, E ) pSEP3::SEP3:GFP reporter line showing SEP3 accumulation in ovules at stage 2-I ( D ) and 2-III ( E ). ( F, G ) pSTK::STK:GFP reporter line in ovules at stage 2-I ( F ) and 2-II ( G ). ( H, I ) pAG::AG:GFP reporter line in ovules at stage 2-I ( H ) and 2-II ( I ). Abbreviations: nu= nucella; ch= chalaza; fu= funiculus; ii= inner integument; oi= outer integument; pl= placenta. Scale bar = 20 μm.

Article Snippet: AG and STK full-length CDS and SPL/NZZ and SEP3Δ shorter versions (SPL/NZZΔ and SEP3Δ respectively) were cloned into Gateway vector GAL4 system (pGBKT7 and pGADT7, Clontech; pTFT1 ).

Techniques: Y2H Assay, Serial Dilution, Hybrid Assay

Journal: Frontiers in Plant Science

Article Title: Functional analysis and interaction networks of Rboh in poplar under abiotic stress

doi: 10.3389/fpls.2025.1553057

Figure Lengend Snippet: Interaction proteins of PyRbohs . (A) Predicted network of protein-protein interactions of PyRbohs using STRING. Spheres represented interacting proteins, with the orange proteins in the center representing PyRboh proteins. The colored lines denoted different types of interaction relationships between proteins: interaction relationships verified by curated databases (blue) or experiments (purple); interaction relationships predicted by gene neighborhood location (green), gene fusion (red), or gene co-occurrence (blue violet); possible interactions of proteins by text mining (olive), co-expression (black), or protein homology (gray-purple). (B) Protein-protein interactions detected using Y2H assays. In Y2H assay, the coding sequences of Poyun17687, Poyun27068 and Poyun21683 was ligated to activation domain vectors (AD, as pGADT7-Poyun17687), and coding sequences of Poyun00939 and Poyun19109 was fused to GAL4 DNA-binding domain vectors, (BD, as pGBKT7-Poyun00939). The Y2H yeast strain (AH109) was used in this assay. The pGADT7 without any coding sequences and pGBKT7-Poyun00939, pGBKT7-19109 were used as the negative control of Y2H. SD/-L-T, SD/-H-L-T, and SD/-A-H-L-T represented SD-Leu-Trp, SD-His-Leu-Trp and SD-Ade-His-Leu-Trp medium. Different concentrations of AbA (0, 400, 800 µg/L) were added for screening.

Article Snippet: The coding sequences of the bait proteins (Poyun00939, Poyun19109) were fused to the GAL4 DNA-binding domain vector (pGBKT7, Clontech, USA), while the coding sequences of the predicted proteins (Poyun270678, Poyun17687, Poyun21683) were fused to the GAL4 activation domain vector (pGADT7, Clontech, USA) ( ).

Techniques: Expressing, Y2H Assay, Activation Assay, Binding Assay, Negative Control