Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of the preparatory and analytical workflows. (A) POMC derived ligands and their downstream signaling cascades. (B) Schematic overview of the thermal proteome profiling (TPP) workflow. MC3R-expressing HEK293 cells were treated with ACTH, α-MSH, or γ-MSH at concentrations of 20, 100, and 500 nM or with DMSO as a vehicle-only negative control. (C) Schematic overview of the TPP data analysis workflow. Protein identification and relative quantification were achieved by direct analysis of the raw LC–MS data, after which various bioinformatics tools were used to infer changes in transcription factor (TF) activity, perform enriched pathway analysis, and identify thermally affected proteins.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Derivative Assay, Expressing, Negative Control, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of identified proteins and thermally stabilized or destabilized proteins. (A) Venn diagrams showing the numbers of proteins exhibiting altered melting points, associations with enriched pathways, and phosphorylation in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH. (B) Venn diagrams showing the numbers of stabilized, destabilized, and phosphorylated proteins after incubation with ACTH, α-MSH, and γ-MSH. (C) Upset plot representing individual numbers of stabilized and destabilized proteins for each ligand and those common between various combinations of ligands.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Phospho-proteomics, Expressing, Incubation

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Characterization of transcription factors. (A) Heat map showing the relative abundance (compared to vehicle-only controls) of the transcription factors CCAR2, HMGB2, DDX21, SRSF7, and TET2 in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH at different ligand concentrations and temperatures. (B) Phosphorylation of tryptic peptides derived from the thermally stabilized and destabilized transcription factors shown in panel A whose activity was inferred to change following stimulation with ACTH, α-MSH, or γ-MSH. Phosphorylation sites are indicated by asterisks next to the modified amino acid (shown in parentheses when the exact amino acid is unknown). (C) Transcription factor activities and relational networks inferred from differential expression data using BITFAM. The heatmap shows fold changes in transcription factor activities (relative to vehicle-only treatments) in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, or γ-MSH. (D) Network showing the interconnectivity of the transcription factors identified within our experimental LC–MS data set.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Expressing, Incubation, Phospho-proteomics, Derivative Assay, Activity Assay, Modification, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Biomolecules

Article Title: Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems

doi: 10.3390/biom11101448

Figure Lengend Snippet: The Uniprot Accession No. of each SFK and expression vector for subcloning.

Article Snippet: The 293 (human embryonic kidney) cell line and cDNAs encoding human Src (clone IRAL047C19), Yes (clone W01A026C12), Fyn (clone W01A056G21), Lck (clone W01A107I15), Hck (clone IRAL034D12), Blk (clone W01A026C12), and Lyn (clone Lyn/pLY30) were obtained from RIKEN BioResource Research Center (RIKEN BRC, Tsukuba, Japan). cDNA encoding human Fgr (clone pF1KB9941), the T N T SP6 Quick Coupled Transcription/Translation System, the T N T T7 Insect Cell Extract Protein Expression System, the T N T SP6 High-Yield Wheat Germ Protein Expression System, the S30 T7 High-Yield Protein Expression System, pF25A ICE T7 Flexi Vector, and pSP64 poly(A) vector were purchased from Promega Corp. (Madison, WI, USA).

Techniques: Expressing, Plasmid Preparation, Subcloning

Journal: Biomolecules

Article Title: Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems

doi: 10.3390/biom11101448

Figure Lengend Snippet: Summary of the kinase activity of SFKs expressed in cell-free protein expression systems, 293 cells, and E. coli BL21(DE3). + or − means presence or absence of phosphorylation activity toward GST-Srctide, respectively.

Article Snippet: The 293 (human embryonic kidney) cell line and cDNAs encoding human Src (clone IRAL047C19), Yes (clone W01A026C12), Fyn (clone W01A056G21), Lck (clone W01A107I15), Hck (clone IRAL034D12), Blk (clone W01A026C12), and Lyn (clone Lyn/pLY30) were obtained from RIKEN BioResource Research Center (RIKEN BRC, Tsukuba, Japan). cDNA encoding human Fgr (clone pF1KB9941), the T N T SP6 Quick Coupled Transcription/Translation System, the T N T T7 Insect Cell Extract Protein Expression System, the T N T SP6 High-Yield Wheat Germ Protein Expression System, the S30 T7 High-Yield Protein Expression System, pF25A ICE T7 Flexi Vector, and pSP64 poly(A) vector were purchased from Promega Corp. (Madison, WI, USA).

Techniques: Activity Assay, Expressing, Phospho-proteomics

Journal:

Article Title: 111 Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo

doi: 10.1111/j.1365-2249.2004.02578.x

Figure Lengend Snippet: Cultured gut-derived T cells show strong adhesion to MAdCAM-1. The adhesion was inhibited by integrin α4 antibody (1 µg/5 × 105 cells) and to a lesser extent by the commercially available MAdCAM-1 antibody (0·1 µg/well) not raised against the specific recombinant MAdCAM-1 protein applied in this assay. The integrin α4β7-negative cell line K562 was arbitrarily assigned a binding index value of 1. Results are expressed as mean of quadriplicate experiments in the same five gut-derived T cell cultures used in the in vivo studies.

Article Snippet: Dr S. Fong, Genentech, Inc. (San Francisco, CA, USA) kindly provided human 293 embryonic kidney cells stably secreting recombinant soluble human MAdCAM-IgG fusion protein.

Techniques: Cell Culture, Derivative Assay, Recombinant, Binding Assay, In Vivo