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: Frontiers in Veterinary Science

Article Title: Case Report: Anti-GABA A Receptor Encephalitis in a Dog

doi: 10.3389/fvets.2022.886711

Figure Lengend Snippet: Antibody diagnostics. Immunofluorescence studies: diluted patient material is incubated with mouse brain sections or human embryonic kidney (HEK) cells transfected with the antigens of interest. Binding of patient antibodies to the respective matrix is visualized by a secondary anti-human/canine antibody coupled with an immunofluorescence dye resulting in a red signal. (A–C) Tissue-based assays, mouse cerebellum. (D) cell-based assay with HEK cells which were transfected with the γ-aminobutyric acid (GABA)-A receptor (GABA A R) subunits α1β3 (all assays from Euroimmun, Lübeck, Germany). Molecular layer (ML), purkinje cell layer (Pu), granular cell layer (GL), white matter (WM). (A) Human antibodies against the GABA A R. (B) Human antibodies against the GABA B R. (C) The canine antibodies against the GABA A R. Please note that the binding pattern is similar to (A) and not to (B) . (D) The canine serum binds to GABA A R transfected HEK cells. Nuclear counterstaining with Hoechst 33342 in blue. Bar in (A) , valid also for (B,C) : 100 μm. Bar in (D) : 25 μm.

Article Snippet: An extensive antibody profile demonstrated a neuropil staining pattern on a tissue-based assay compatible with GABA A R antibodies ( ); the diagnosis was confirmed by binding of serum and CSF to GABA A R (α1β3 subunits) transfected Human Embryonic Kidney cells [all assays from Euroimmun, Lübeck, Germany, used with in-house-protocols; for the canine material, secondary anti-dog Immunoglobulin G (IgG) was used].

Techniques: Immunofluorescence, Incubation, Transfection, Binding Assay, Cell Based Assay