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:

Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

doi: 10.1073/pnas.0402889101

Figure Lengend Snippet: Stable cell line expresses elevated SUMO and sumoylated proteins. HEK 293 Tet-On cells were transfected with either pTRE2hyg2-Myc-SUMO or pTRE2hyg2-Myc-Luc vector. Cells stably expressing Myc-SUMO and Myc-Luc were selected with 300 μg/ml hygromycin. After a 48-h incubation with or without 2 μg/ml Dox, whole-cell extracts were resolved by using NuPage gels and probed with anti-Myc (A), anti-SUMO (B), anti-p53 (C), or anti-β-actin (D) antibody. The p53 is marked with a single asterisk, and the sumoylated p53 is marked with double asterisks.

Article Snippet: Human embryonic kidney 293 Tet-On cell line, pTRE2hyg2-Myc, pTRE2hyg2-Myc-luciferase (Luc), hygromycin, and doxycycline (Dox) were purchased from BD Biosciences/Clontech.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Incubation

Journal:

Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

doi: 10.1073/pnas.0402889101

Figure Lengend Snippet: Myc-tagged SUMO and sumoylated proteins mainly localized in the nucleus. After Myc-SUMO stably expressed HEK 293 Tet-On cells were induced with 2 μg/ml Dox for 48 h, cells were fixed with 3.7% formaldehyde and stained with anti-Myc primary antibody and FITC-conjugated secondary antibody. DAPI was used to stain nuclei.

Article Snippet: Human embryonic kidney 293 Tet-On cell line, pTRE2hyg2-Myc, pTRE2hyg2-Myc-luciferase (Luc), hygromycin, and doxycycline (Dox) were purchased from BD Biosciences/Clontech.

Techniques: Stable Transfection, Staining

Journal:

Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

doi: 10.1073/pnas.0402889101

Figure Lengend Snippet: Cellular sumoylated proteins show a similar pattern to in vitro isolated sumoylated proteins in 2D gel electrophoresis. Whole cell extracts from HEK 293 Tet-On cells expressing Myc-SUMO were resolved by using 7-cm 2D gel and probed with anti-Myc antibody (A). The Myc-SUMO is marked with an arrow. The in vitro isolated sumoylated proteins were resolved by using 18-cm 2D gel and silver stained (B). Biotin–SUMO is also marked with an arrow.

Article Snippet: Human embryonic kidney 293 Tet-On cell line, pTRE2hyg2-Myc, pTRE2hyg2-Myc-luciferase (Luc), hygromycin, and doxycycline (Dox) were purchased from BD Biosciences/Clontech.

Techniques: In Vitro, Isolation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Expressing, Staining

Journal:

Article Title: Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis

doi: 10.1073/pnas.0402889101

Figure Lengend Snippet: hnRNP A1, hnRNP F, and hnRNP K are sumoylated. The whole-cell extracts from HEK 293 Tet-On cells expressing Myc-SUMO or Myc-Luc were immunoprecipitated with either anti-Myc (A), anti-hnRNP F (B), or hnRNP K (C) antibody. The immunoprecipitates were resolved by NuPage gels and probed with anti-hnRNP A1 (A) or anti-Myc (B and C) antibody. Sumoylated hnRNP proteins are marked with arrows. The lower bands in B are caused by IgG heavy chain cross-reacting with the secondary antibody.

Article Snippet: Human embryonic kidney 293 Tet-On cell line, pTRE2hyg2-Myc, pTRE2hyg2-Myc-luciferase (Luc), hygromycin, and doxycycline (Dox) were purchased from BD Biosciences/Clontech.

Techniques: Expressing, Immunoprecipitation

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