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: PLoS ONE

Article Title: Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

doi: 10.1371/journal.pone.0116412

Figure Lengend Snippet: (A) 2.5% agarose gel electrophoresis of RT-PCR products derived from isolated mRNA after 293T cells transfection with obtained plasmids. Lanes 1–3, PCR fragments of 435 bp correspond to P1, P2, and P3 plasmids; Lanes 4–6, PCR fragments of 624 bp correspond to P1, P2, and P3 plasmids; K, negative control sample (total mRNA extracted from transfected cells); M, 100bp DNA Ladder (SibEnzyme, Russia). (B) Western blot analysis of expression products of genes encoding TCI-N, TCI-N2, and TCI-N3 proteins in transfected 293T cells. Lanes P1, P2, P3 are lysates of 293T cell transfected with P1, P2, P3, respectively; lane К represents lysates of 293T cells transfected with vector plasmid pcDNA3.1. The membrane containing the cell-associated fractions was also probed with аnti-beta аctin antibody to control for equal loading of the samples. (C) Flow cytometry analyses of expression products of target genes after transfection of 293T cells with P1, P2, and P3 plasmids. Histogram overlays of target proteins stained with FITC-labeled 29F2 monoclonal antibodies (green peak, TCI-N; blue peak, TCI-N2; and yellow peak, TCI-N3 polyepitope protein expression). Negative control (read peak) represents 293T cells transfected with vector plasmid pcDNA3.1.

Article Snippet: 293T (human embryonic kidney cell line) cells were cultured in RPMI (Biolot, Russia) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Rockville, MD).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Isolation, Transfection, Negative Control, Western Blot, Expressing, Plasmid Preparation, Membrane, Control, Flow Cytometry, Staining, Labeling, Bioprocessing

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: eLife

Article Title: The angiopoietin-like protein ANGPTL4 catalyzes unfolding of the hydrolase domain in lipoprotein lipase and the endothelial membrane protein GPIHBP1 counteracts this unfolding

doi: 10.7554/eLife.20958

Figure Lengend Snippet: Panel A shows a sequence alignment of the first 55 residues of fully processed human ANGPTL3, ANGPTL4, and ANGPTL8. Identical sequences are highlighted by blue letters. The filled red box highlights the two-residue acidic motif at the start of the N-terminal α-helix, followed by a conserved α-helical region (open box). The two cysteine residues unique to ANGPTL4 are shown in yellow boxes. Panel B shows the time-dependent unfolding of 10 µM LPL by 1 µM ANGPTL3 ( green circles ) as defined by the appearance of bimodality in the isotope envelopes for peptide 131–165. For comparison the unfolding of LPL by 1 µM ANGPTL4 1–159 is shown as solid (wild-type) and hatched (E15K) gray lines. Spontaneous unfolding is shown by black triangles. Panel C shows the residual lipolytic activity of 10 µM LPL incubated for 10 min alone or in the presence of 1 µM ANGPTL3, 1 µM ANGPTL3 and 30 µM GPIHBP1, or 1 µM ANGPTL4 1–159 . DOI: http://dx.doi.org/10.7554/eLife.20958.012

Article Snippet: Full-length ANGPTL3 produced in HEK293 cells was purchased from ProSpec (Ness-Ziona, Israel).

Techniques: Sequencing, Residue, Comparison, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Aquaporin7 plays a crucial role in tolerance to hyperosmotic stress and in the survival of oocytes during cryopreservation

doi: 10.1038/srep17741

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of AQP7 expression in mouse oocytes in the presence of 0.25 M, 0.5 M, 0.75 M and 1 M sucrose, respectively. ( B ) Summary data of the immunofluorescence analysis (n ≥ 5). ( C ) Immunofluorescence analysis of AQP7 and F-actin colocalization in mouse oocytes (red: AQP7; green: F-actin). Scale bar ( A – C ), 20 μm, ( D ) Co-immunoprecipitation of AQP7 and F-actin. IP, immunoprecipitation. ( E ) Green fluorescent protein (GFP) intensity in 293FT cells transfected with the GFP-hAQP7 fusion protein expression plasmid or with the GFP vector alone in the presence of 8% EG, 9.5% DMSO, and 0.5 M sucrose, respectively. Scale bar, 10 μm. ( F ) Summary data of the immunofluorescence analysis (calculated for 20 cells for each condition obtained from three independent experiments). Data are presented as the mean ± SE, ** P < 0.01 compared to the corresponding control.

Article Snippet: The human embryonic kidney cell line (293FT) was purchased from ScienCell (Carlsbad, USA), and cultured in DMEM (Gibco, Grand Island, USA) containing 10% fetal bovine serum (Gibco).

Techniques: Immunofluorescence, Expressing, Immunoprecipitation, Transfection, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Aquaporin7 plays a crucial role in tolerance to hyperosmotic stress and in the survival of oocytes during cryopreservation

doi: 10.1038/srep17741

Figure Lengend Snippet: ( A ) Oocytes were pretreated with LY294002, staurosporine, U0126 and SP600125, respectively, and the immunofluorescence intensities of AQP7 were analysed in mouse oocytes in the presence of 8% EG. Scale bar, 20 μm. ( B ) Summary data of the immunofluorescence analysis (n ≥ 7). ( C ) 293FT cells were transfected with the GFP-hAQP7 fusion protein expression plasmid and treated as in ( A ). The immunofluorescence intensities of GFP-hAQP7 were analysed in the presence of 8% EG. Scale bar, 10 μm. ( D ) Summary data of the immunofluorescence analysis (calculated for 20 cells for each condition obtained from three independent experiments). ( E ) Western blotting analysis of GFP-hAQP7 in 293FT cells treated as in ( C ). ( F ) Summary data of the Western blotting analysis (n = 3). Data are presented as the mean ± SE. ** P < 0.01 compared to the corresponding control.

Article Snippet: The human embryonic kidney cell line (293FT) was purchased from ScienCell (Carlsbad, USA), and cultured in DMEM (Gibco, Grand Island, USA) containing 10% fetal bovine serum (Gibco).

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Western Blot, Control