grb2 gene  (Addgene inc)

Journal: Veterinary Sciences

Article Title: VEGF-B , VEGF-A , FLT-1 , KDR , ERBB2 , EGFR , GRB2 , RAC1 , CDH1 and HYAL-1 Genes Expression Analysis in Canine Mammary Gland Tumors and the Association with Tumor ClinicoPathological Parameters and Dog Breed Assessment

doi: 10.3390/vetsci8100212

Figure Lengend Snippet: Expression levels of 10 analyzed genes ( VEGF-B , VEGF-A , FLT-1 , KDR , ERBB2 , EGFR , GRB2 , RAC1 , CDH1 and HYAL-1 ) in canine mammary tumors (CMTs) and tumor adjacent tissues in all studied dogs. qRT-PCR data are represented as delta Ct values. Dots indicate outliers. * p < 0.05 measured with Wilcoxon or t -tests.

Article Snippet: The expression level of genes were measured using TaqMan TM Gene Assays with FAM dye ( VEGF-B Cf02721109_u1, VEGF-A Cf02674018_m1, FLT-1 Cf02696454_g1, KDR Cf02627749_m1, ERBB2 Cf02621873_g1, EGFR Cf02626541_m1, GRB2 Cf02667172_m1, RAC1 Cf02699525_m1, CDH1 Cf02624268_m1, HYAL-1 Cf02718719_s1) and TaqMan Universal Master Mix (Applied Biosystems, Carlsbad, CA, USA) on the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer‘s protocol.

Techniques: Expressing, Quantitative RT-PCR

Journal: Veterinary Sciences

Article Title: VEGF-B , VEGF-A , FLT-1 , KDR , ERBB2 , EGFR , GRB2 , RAC1 , CDH1 and HYAL-1 Genes Expression Analysis in Canine Mammary Gland Tumors and the Association with Tumor ClinicoPathological Parameters and Dog Breed Assessment

doi: 10.3390/vetsci8100212

Figure Lengend Snippet: Expression levels of analyzed genes ( VEGF-B , FLT-1 , EGFR , GRB2 and ERBB2 ), considering clinical parameters in CMTs and tumor adjacent tissues of different dog breeds. Expression levels of VEGF-B , and FLT genes in metastasis-free carcinoma samples ( A , B ), VEGF-B in groups with carcinoma stage I ( C ), EGFR in group with grade I ( G ), VEGF-B , GRB2 and ERBB2 in groups with carcinoma stage III ( E –G). Dots indicate outliers. * p < 0.05, ** p < 0.01 measured with Wilcoxon or t -tests.

Article Snippet: The expression level of genes were measured using TaqMan TM Gene Assays with FAM dye ( VEGF-B Cf02721109_u1, VEGF-A Cf02674018_m1, FLT-1 Cf02696454_g1, KDR Cf02627749_m1, ERBB2 Cf02621873_g1, EGFR Cf02626541_m1, GRB2 Cf02667172_m1, RAC1 Cf02699525_m1, CDH1 Cf02624268_m1, HYAL-1 Cf02718719_s1) and TaqMan Universal Master Mix (Applied Biosystems, Carlsbad, CA, USA) on the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer‘s protocol.

Techniques: Expressing

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a. ddPCA uses two protein fragment complementation (PCA) selection assays to quantify the effects of mutations on the abundance ( abundancePCA ) of a protein of interest A and its binding to an interaction partner B ( bindingPCA ) based on growth in the presence of methotrexate. b. Reproducibility of fitness estimates from ddPCA for single and double AA substitutions between all biological replicates. Pearson correlations are indicated in red. c. Comparison of individually measured growth rates (slope of a linear fit of the log 10 (OD 600nm ) against time during the exponential phase) of GRB2-SH3 variants to growth rates inferred from deep sequencing data (see Methods). The dashed line corresponds to the linear regression model. R=Pearson correlation. d. 3D structures of the SH3 domain of GRB2 bound to the ligand peptide of GAB2 (PDB entry 2VWF) and the third PDZ domain of PSD95 bound to the ligand peptide of CRIPT (PDB entry 1BE9). e. Fitness density distributions. Total variant counts for singles (red) and doubles (blue) are indicated (purple indicates overlapping density distributions). Vertical dashed lines indicate the median fitness of STOP codon mutations in the central 50% of the coding sequence. f-g. Heatmaps of the fitness effects of single AA substitutions for GRB2-SH3 ( f ) and PSD95-PDZ3 ( g ) corresponding to the bindingPCA (upper panel) and abundancePCA (lower panel) assays. The final row in each heatmap indicates the minimal distance between domain and ligand side chain heavy atoms (or alpha carbon atoms in the case of glycine). Amino acid labels are coloured in red or blue for positive or negatively charged residues respectively, green for aromatic amino acids and highlighted in yellow for hydrophobic residues. Fitness values more extreme than ±1.5 were set to this limit. h. Scatterplots comparing abundance and binding fitness of single AA substitutions. Variants are coloured by the corresponding residue position in the domain structure: core (relative solvent accessible surface area, RSASA<0.25), surface (RSASA≥0.25) or ligand binding interface (minimal side chain heavy atom distance<5Å).

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: Selection, Binding Assay, Sequencing, Variant Assay, Ligand Binding Assay

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a-b. Heatmaps showing inferred changes in free energies of binding and folding for GRB2-SH3 ( a ) and PSD95-PDZ3 ( b). The final row in each heatmap indicates the ligand distance and amino acid labels are coloured according to the chemical structure and properties of their side-chains (see ). Free energy changes of ligand-proximal residues (ligand distance<5Å) are boxed and asterisks indicate major allosteric positions. Lower confidence estimates are indicated with dots (95% confidence interval ≥1kcal/mol). Free energy changes more extreme than ±2.5kcal/mol were set to this limit. c. Scatterplots comparing confident binding and folding free energy changes of mutations in the core, surface and binding interface. Contours indicate estimates of 2D densities using 6 contour bins. Axis limits were adjusted to include the largest contour bin (more extreme data points are not shown). d. Distributions of confident binding (red) and folding (blue) free energy changes. X-axis limits were adjusted to match those in panel c. e. Percentage of mutations that significantly decrease (top) or increase (bottom) fitness in the binding assay (FDR=0.05) categorized by their biophysical mechanism. Pleiotropic mutations have significant changes in free energies of both folding and binding (FDR=0.05) and are classified as either synergistic or antagonistic depending on whether their effects are in the same or different direction respectively. See Figure S7 for the GB1 domain.

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a. 3D structures of the GRB2-SH3 and PSD95-PDZ3 domains where residue atoms are colored by the position-wise average change in the free energy of folding. Ligands are shown as black sticks. b. Violin plots indicating distributions of confident changes in free energy of folding stratified by position in the structure (two-sided Mann-Whitney U test p-value<2.2e-16 comparing mutations in the core versus the remainder for both protein domains). c. Negative correlation between the position-wise average change in free energy of folding and the solvent exposure of the corresponding residue (RSASA). R = Pearson correlation. d. Percentage of core, surface or binding interface residues shown separately for de-stabilising residues (positions with ≥5 stabilizing mutations, folding ΔΔG<0, FDR=0.05) and the remainder. Inset numbers are total counts. See Figure S9 for the GB1 domain.

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: MANN-WHITNEY, Binding Assay

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a. 3D structures of the GRB2-SH3 and PSD95-PDZ3 domains where residue atoms are colored by the position-wise average absolute change in the free energy of binding. Ligands are shown as black sticks. b. Domain structures with orthosteric sites (ligand distance<5Å) highlighted in red spheres and major allosteric site residues highlighted in orange spheres. c. Relationship between the position-wise average absolute change in free energy of binding and the distance to the ligand (minimal side chain heavy atom distance). Major allosteric sites (orange) are defined as non-binding interface residues with weighted average absolute change in free energy of binding higher than the average of binding interface residue mutations (red). Class-switching residues in PSD95-PDZ3 are those that favour a change in specificity for a T-2F ligand defined in McLaughlin et al. 2012 . See Figure S12 for the GB1 domain.

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: Binding Assay

Journal: ACS Omega

Article Title: Lipid Raft Phase Modulation by Membrane-Anchored Proteins with Inherent Phase Separation Properties

doi: 10.1021/acsomega.9b00327

Figure Lengend Snippet: Three-component phase-separating proteins on the phase-separated lipid membrane. (a) PRM × 4, GRB2-GST, and LAT sequence peptide were added from solution to interact with ternary mixture GUVs with phase separation properties (green and gray for separated domains). LAT sequence peptide (green) was labeled with Atto488 at the C-terminal and had a polyhistidine tag at N-terminal, so it spontaneously anchored itself to the membranes by strongly binding to Ni-NTA lipid. (b–g) Matching typical images of the Atto488 protein channel and the TexasRed-DHPE lipid channel of GUVs with multiple-phase domains. Scale bars are 5 μm. Lipid compositions were 42% DOPC, 29% DPPC, 14% cholesterol, 5% DOPS, 10% Ni-DGS, 0.1% TR-DHPE.

Article Snippet: The GRB2 gene was obtained from AddGene, which was deposited by Vale lab. GRB2 plasmid-introduced E. coli was grown at 37 °C for 3–5 h to overexpress by IPTG 0.5 mM RT overnight.

Techniques: Membrane, Targeted Proteomics, Sequencing, Labeling, Binding Assay