Journal: Frontiers in Plant Science

Article Title: A Cryophyte Transcription Factor, CbABF1, Confers Freezing, and Drought Tolerance in Tobacco

doi: 10.3389/fpls.2019.00699

Figure Lengend Snippet: CbABF1 subcellular localization and transactivation activity assays. (A) Localization of p35S:CbABF1-YFP in tobacco leaves, DAPI staining was used to indicate the nuclei. Bars = 15 μm. (B) A schematic diagram of the CbABF1 protein sequence and its truncated fragments that were fused to the GAL4 DNA-binding domain expressed from the yeast vector pGBKT7. The bZIP domain and four conserved domains are indicated. (C) Transactivation analysis of CbABF1 in yeast. The constructs for expression of the GAL4 DNA-binding domain fused with CbABF1, CbABF1Δ1, and CbABF1Δ2 were co-transformed with empty pGADT7 into yeast strain AH109, respectively. A 10-fold dilution series (10 -1 to 10 -3 ) of transformants was spotted on SD medium without Leu and Trp (–LW), or without Leu, Trp, and His (–LWH) plus 35 mM 3-amino-1,2,4-triazole (3-AT). The plates were incubated at 30°C for 4 days and subjected to the X-gal assay. Empty pGBKT7 with empty pGADT7 was used as the negative control.

Article Snippet: The purified double-stranded cDNA was inserted into a Sma I-linearized pGADT7-Rec vector (3 μg) with T4 DNA ligase (New England Biolabs, Ipswich, MA, United States).

Techniques: Activity Assay, Staining, Sequencing, Binding Assay, Plasmid Preparation, Construct, Expressing, Transformation Assay, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Cathepsin D interacts with adenosine A 2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

doi: 10.1016/j.jbc.2022.101888

Figure Lengend Snippet: Validation of the interaction between CtsD and the A 2A R C-terminal tail in yeast. A , transformation of Yeast 2H Gold strain with pGBKT7, pGBKT7-A 2A R 284–410 (bait), pGADT7 and pGADT7-CtsD 321–410 (prey) vectors in a pairwise combination, according to the numbered yeast line. The yeast cells were grown on SD/Leu − Trp − or SD/Leu − Trp − Ade − His − (in the presence of Aureobasidin and X-α-Gal) selective medium. pGBKT7-p53 and pGADT7-SV40 small T antigen were used as positive controls and pGBKT7-p53 and pGADT7-Lamin expressing vectors were used as negative controls. B , presentation of the different maturation forms of CtsD. Characterization of A 2A R 284–410 interactor clones that encode the different ORFs of the CtsD. The amino acid numbers of pre–pro-CtsD protein (P18242) is based on UniProtKB/Swiss-Prot databases. S: N-terminal secretion signal peptide (20 amino acids [AA]); P: propeptide (44 AA); C: cleavage site between the light and heavy chain of CtsD; arrows indicate the processing sites; D: catalytic Asp residues (D97 and D295) are depicted by black triangles ; N: N-linked glycosylation sites at Asn residues (N134 and N261) are depicted by empty triangles . A 2A R, adenosine A 2A receptor; CtsD, cathepsin D.

Article Snippet: Nontransformed Y187 yeast cells were used as a negative control, and 25 ng SV40 large T PCR product with 0.5 μg pGADT7-Rec vector (catalog no.: 634901; Takara-Clontech) were cotransformed into Y187 yeast cells as a positive control.

Techniques: Transformation Assay, Expressing, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: BplMYB46 from Betula platyphylla Can Form Homodimers and Heterodimers and Is Involved in Salt and Osmotic Stresses

doi: 10.3390/ijms20051171

Figure Lengend Snippet: Homodimeric and heterodimeric analysis of BplMYBs using a yeast two-hybrid assay (Y2H). ( A ) BplMYB46 was cloned into a pGADT7-Rec vector (AD-prey) and interacted separately with the eight MYB proteins, which were fused to the GAL4 DNA binding domain in the yeast pGBKT7 vector (BD-baits). Empty AD-prey and each BD-bait were used as negative controls; AD-BplMYB46-prey and empty BD-bait were used as negative controls; and AD-T-prey and BD-53-bait were used as positive controls. ( B ) BplMYB46-N (without its activation regions) was cloned into pGBKT7 vector (bait) and interacted with itself and with the other eight MYB proteins cloned into the pGADT7-Rec vector (preys). Empty BD-bait and each AD-prey were used as negative controls; BD-BplMYB46-N-bait and empty AD-prey were used as negative controls; BD-53-bait and AD-T-prey were used as positive controls. The yeast cells were grown on SD/-Trp/-Leu (double dropout (DDO)) and selective dropout media: SD/-Trp/-Leu/-His/-Ade/X-α-Gal (quadruple dropout (QDO)/X-α-Gal).

Article Snippet: The open reading frame (ORF) of the BplMYB46 cDNA, without the terminal codon, was inserted into the pGADT7-Rec vector via its SmaI (Promega, Madison, WI, USA) site as the prey construct.

Techniques: Y2H Assay, Clone Assay, Plasmid Preparation, Binding Assay, Activation Assay