Journal: Cell reports

Article Title: Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS 1 )

doi: 10.1016/j.celrep.2023.112015

Figure Lengend Snippet:

Article Snippet: Plasmid pDS11-SacB , Genscript , N/A.

Techniques: Virus, Recombinant, Functional Assay, Protease Inhibitor, Produced, Plasmid Preparation, Software, Electroporation, Fluorescence, Flow Cytometry, Chromatography, Affinity Column, Size-exclusion Chromatography, Purification

Journal: Journal of biomolecular NMR

Article Title: Tuning a timing device that regulates lateral root development in rice

doi: 10.1007/s10858-019-00258-0

Figure Lengend Snippet: a) Sequence alignment of LRT2 with cyclophilins from wheat (TaCypA), human (CypA) and E. coli (PPIB). b) The LRT2 homology model (green) aligned with the crystal structure of human Cyclophilin A (cyan, PDB:1awr.1) shows the conserved catalytic residues H61, R62, Q118, F120, W128 and H133 (LRT2 numbering). c) Residues in the loop preceding W121 in hCypA that are not conserved in LRT2. d) Corresponding residues in the loop preceding W128 in the LRT2 homology model

Article Snippet: Custom vectors for bacterial expression of PPIB (Uniprot entry {"type":"entrez-protein","attrs":{"text":"P23869","term_id":"2507227","term_text":"P23869"}} P23869 ) and TaCypA (Uniprot entry {"type":"entrez-protein","attrs":{"text":"Q93W25","term_id":"75305737","term_text":"Q93W25"}} Q93W25 ) were purchased from GenScript (Piscataway, NJ).

Techniques: Sequencing

Journal: Journal of biomolecular NMR

Article Title: Tuning a timing device that regulates lateral root development in rice

doi: 10.1007/s10858-019-00258-0

Figure Lengend Snippet: Isomerization rates for LRT2 mutants and homologs determined by 1 H- 1 H ROESY and 1 H- 15 N ZZ exchange experiments.

Article Snippet: Custom vectors for bacterial expression of PPIB (Uniprot entry {"type":"entrez-protein","attrs":{"text":"P23869","term_id":"2507227","term_text":"P23869"}} P23869 ) and TaCypA (Uniprot entry {"type":"entrez-protein","attrs":{"text":"Q93W25","term_id":"75305737","term_text":"Q93W25"}} Q93W25 ) were purchased from GenScript (Piscataway, NJ).

Techniques:

Journal: Journal of biomolecular NMR

Article Title: Tuning a timing device that regulates lateral root development in rice

doi: 10.1007/s10858-019-00258-0

Figure Lengend Snippet: (a-c) Expanded region of the 1H-15N NZZ exchange NMR spectrum (mixing time = 0.55s) of 15N-OsIAA1172−125 in the presence of hCypA (a) or PPIB from E. coli (b), and of the ROESY spectrum (70 ms) of OsIAA1198−109 in the presence of TaCypA (c). Exchange peaks between the 104W-NHεcis and 104W-NHεtrans auto peaks are observed in the presence of each homolog. (d-f) Fitting (lines) of normalized peak intensities for the auto (Itt and Icc) and exchange (Itc and Ict) peaks in the presence of hCypA (d), PPIB (e) and TaCypA (f) yielded the corresponding kexobs values (Table 2)

Article Snippet: Custom vectors for bacterial expression of PPIB (Uniprot entry {"type":"entrez-protein","attrs":{"text":"P23869","term_id":"2507227","term_text":"P23869"}} P23869 ) and TaCypA (Uniprot entry {"type":"entrez-protein","attrs":{"text":"Q93W25","term_id":"75305737","term_text":"Q93W25"}} Q93W25 ) were purchased from GenScript (Piscataway, NJ).

Techniques:

Journal: bioRxiv

Article Title: Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex

doi: 10.1101/2020.03.04.976134

Figure Lengend Snippet: A. Design schematic of the approach utilized to create the SPARC through a VEGAS assembly approach. Each individual transcriptional unit (TU) was checked to replace promoters or terminators that utilized identical sequences and replaced with an alternative sequence indicated by a purple underline. These TUs were assembled into level 1 plasmids by Golden Gate reaction. Subsequently, they were amplified by PCR using primers specific for the Vegas Adaptor (VA) sequences specific for their TU cassette. In example, for the first Repressor/Substrate TU the TU was amplified using primers for VA1 and VA3 and purified by a PCR cleanup column (NEB). The acceptor plasmid was cut with EcoRI and both TU and acceptor plasmid was transformed into yeast and recombinant plasmids were selected on synthetic drop out (SDO) plates lacking Leucine. B. Primary SPARC transformants were struck out onto fresh SDO -Leu and imaged for Venus expression, demonstrating varying levels of reporter expression that correlate with TPL repressor domains. Plasmid DNA was purified from these strains and sequenced to confirm the proper recombination of TU cassettes. C. Time course flow cytometry of SPARC strains following auxin addition. For all cytometry experiments, the indicated TPL construct is fused to IAA14, because this IAA works better in haploid yeast strains that IAA3. Every point represents the average fluorescence of 5-10,000 individually measured yeast cells (a.u. - arbitrary units). Auxin (IAA-10μM) was added at the indicated time (gray bar, + Aux). Four independent experiments are shown for each construct. D. The yeast TPL homolog Tup1 and its partner protein Cyc8 do not repress the SPARC. Quantified fluorescence from the single locus auxin response circuit (SPARC) introduced into Tup1 and Cyc8 anchor away lines demonstrates no increased fluorescence from the reporter upon depletion of Tup1 or Cyc8 from the nucleus. Anchor away depletion of Tup1 or Cyc8 results in slower yeast growth. To normalize for this disparity in growth, Venus fluorescence was normalized to red autofluorescence, where each pixel was normalized to the corresponding red autofluorescence collected for that position and plotted as a boxplot. Two individual biological replicates (two separate experiments) were evaluated, and the data was pooled. E-F. Med21 N terminal deletions are viable in Saccharomyces and demonstrate altered SPARC transcriptional states. E. A representative grayscale image of fellow fluorescence of spot plates of yeast strains carrying SPARC plasmids in Med21 N-terminal deletions. Each is plated at an OD600 of 0.1 on SDO with or without auxin (10μM IAA). F. Venus fluorescence from ( E ) was normalized to red background (autofluorescence), where each pixel was normalized to the corresponding red autofluorescence collected for that position and plotted as a boxplot. Two individual biological replicates (two separate experiments) were evaluated, and the data was pooled and is presented as boxplots.

Article Snippet: To create an acceptor plasmid for the assembled transcriptional units, we synthesized a custom vector containing VA1 and VA2 homology sites for recombination (Twist Bioscience, South San Francisco, CA).

Techniques: Sequencing, Amplification, Purification, Plasmid Preparation, Transformation Assay, Recombinant, Expressing, Flow Cytometry, Cytometry, Construct, Fluorescence