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: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Functional analysis of the VNTRs within ABL1 -MS1 in luciferase reporter vector promoter region. a ABL1 promoter vector (p2000) and four promoter vectors in which four different lengths of ABL1 -MS1 (TR13–TR16) were inserted were used. The ABL1 promoter region is indicated by diagonal squares, and the luciferase gene is indicated by gray squares. Four different lengths of ABL1 -MS1 were inserted after the luciferase gene and are indicated by black squares. b The above five different types of promoter vectors were transfected into 293 T and UC3 cells. ABL1 promoter activity was measured by luciferase assay

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Functional Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: VNTR analysis of ABL1 -breakpoint cluster region. a Schematic of the VNTR region of ABL1 . 11 exons are marked as black boxes and 10 introns as white boxes. One VNTR region was identified in the ABL1 -breakpoint cluster region, named MS1, and marked with an asterisk. b The location of ABL1 -MS1, the size of the repeating unit, and the consensus sequence were confirmed from the genome information of NCBI

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Sequencing

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Allelic type patterns of ABL1 -MS1 in cancer-free male controls and bladder cancer patients. a Haplotype patterns in cancer-free controls and cases with bladder cancer. The left panel is the haplotype pattern for ABL1 -MS1 region from control samples. Three genotypes were identified consisting of two different ABL1 -MS1 alleles. The right panel is the ABL1 -MS1 haplotype pattern seen in bladder cancer patients, showing five genotypes consisting of four different ABL1 -MS1 alleles. The first and last lanes correspond to a 100-bp (M1; Invitrogen Co., CA, USA) and a 1-kb size marker (M2; Invitrogen Co.). b Frequency of genotypes between controls and bladder cancer cases. N corresponds to the total number of samples tested for the allele of ABL1 -MS1. C corresponds to the common alleles (13TR, 15TR) and indicates alleles with a frequency of 1% or more. Rare alleles (R) with a frequency of less than 1% correspond to 14TR and 16TR. * Statistically significant ( P < 0.05)

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Control, Marker

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Comparison of allelic frequency of ABL1 -MS1 between controls and bladder cancer

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Comparison

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Meiotic segregation of ABL1 -MS1. a Meiotic inheritance of the ABL1 -MS1 in a third-generation family. ABL1 -MS1 were analyzed for minisatellite length in genomic DNA from family members. The pedigree demonstrates the relationship between family groups used in this study: first-generation (lanes 1 and 2, grandfather and grandmother, respectively); second-generation (lanes 3 and 4, father and mother); and third-generation (lanes 5 and 6, children from parents 3 and 4. b Meiotic inheritance the ABL1 -MS1 in three of second-generation. The first-generation is denoted as 1 and 2 (mother and father). The second-generation was shown as 3 and 4, and 5 (children). M corresponds to the size marker

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Marker

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Composition of repeats unit of ABL1 -MS1 alleles

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques:

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