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MRGPRX2-mediated mast cell activation promotes early inflammatory reaction in psoriasis

ABSTRACT: Total protein of psoriasis skin samples was collected and dilution to 1 ml. 1 μg Recombinant Human GPCR MRGX2 protein (GST tag, Abcam, Cambridge, UK) was added into the protein solution, rotate for 4 h at room temperature. Then the anti-GST magnetic bead (BD, New Jersey, USA) was used for get the binding protein of MRGPRX2. Polypeptide segments were detected by liquid chromatography-mass spectrometry. Moreover, the Targeted Proteomics was used to prove BD1, BD3, LL-37, S00A9 and S100A6 (n=3).

ORGANISM(S): Homo Sapiens

SUBMITTER: Songmei Geng

PROVIDER: PXD051684 | iProX | Tue Apr 23 00:00:00 BST 2024

REPOSITORIES: iProX

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Project description:Total protein of psoriasis patients’ skin samples (n=4) was collected and dilution to 1 ml. 1 μg Recombinant Human GPCR MRGX2 protein (GST tag) was added into the protein solution, rotate for 4 h at room temperature. Then the anti-GST magnetic bead was used for get the binding protein of MRGPRX2. Polypeptide segments were detected by liquid chromatography-mass spectrometry.

2023-12-12 | PXD047735 |

LC-MS/MS proteomic analysis of human tissues

Project description:This project is to identify the presence of bacterial flagellins in human liver samples. Human liver protein lysates were separated on SDS-PAGE gels and visualized with Coomassie stain. The positions at which flagellins were detected using anti-flagellin antibodies from Covalab Inc. (France) and Abcam Inc. (UK) were excised for flagellin identification.

2023-11-21 | PXD041941 | Pride

Project description:GST/GST-WLG3

| PRJNA1314697 | ENA

Expression data from HMC-1 mast cells

Project description:We demonstrate that the G protein Gi3 is the cellular target of the adenosine A3 receptor (A3R). By using a cell permeable peptide comprising the C-terminal end of GÎ±i3 fused to an importation sequence (ALL1) as a selective inhibitor of Gi3 signaling, we show that by coupling to Gi3, the A3R stimulates multiple signaling pathways in human mast cells, leading to upregulation of cytokines, chemokines and growth factors.Following contact with activated T cell membranes, endogenous adenosine binds to and activates the A3R, resulting in Gi3-mediated signaling. Specifically, the majority of ERK1/2 signaling initiated by contact with activated T cell membranes, is mediated by Gi3, giving rise to ALL1-inhibitable cellular responses. These results unveil the physiological GPCR that couples to Gi3 and establish the important role played by this G-protein in inflammatory conditions that involve adenosine-activated mast cells. We used microarrays to detail the effect of ALL1 on gene expression of HMC-1 cells activated directly by the A3 receptor, or by contact with activated T cell membranes. The experiment consisted of 6 treatments in duplicates, a total of 12 samples. The cells were activated via two different activation pathways (A3 and T membranes), with or without pretreatment with the drug ALL1.

2010-04-12 | E-GEOD-19888 | biostudies-arrayexpress

Interactome analysis of human Rab11a with proteins from influenza A virus infected cell lysates

Project description:The aim of this project was to determine which viral and host proteins bind to purified recombinant human GTPase Rab11a from lysates of HEK-293T cells infected with influenza A/WSN/33 (H1N1) virus. To this aim we used a GST pull-down assay. Approximately 8∙106 HEK-293T cells were infected with influenza A/WSN/33 (H1N1) virus at a multiplicity of infection of 5 (MOI=5) or mock infected. Twelve hours (h) post-infection cells were harvested by centrifugation at 1,000 rpm for 5 min and lysed for 1 h in lysis buffer [50 mM HEPES-NaOH pH 7.5, 200 mM NaCl, 0.5 % (v/v) IGEPAL CA-630 (Sigma-Aldrich), 1 mM β-mercaptoethanol, 1x PMSF, 1x protease inhibitor (Roche, complete mini, EDTA-free)] at 4 °C. Supernatants were collected by centrifugation at 13,000 rpm for 5 min at 4 °C. Approximately 0.2 mg of purified GST-tagged Rab11CA (constitutively active Rab11a with a Q70L substitution) or GST tag was bound to pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) (100 µl slurry per 1 ml sample volume) at 4 °C for 3 h in binding buffer [50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 10 % (v/v) glycerol, 8 mM MgCl2, 10 µM GTP-γ-S (Abcam)]. GST-Rab11CA or GST tag bound beads were incubated with virus infected or mock infected cell lysates for 1 h at 4 °C followed by five washes in binding buffer. Rab11CA and bound proteins were released in the same buffer supplemented with 1 mM DTT and 0.1 mg human rhinovirus (HRV) 3C protease to cleave off Rab11CA from the GST tag. After 1 h incubation at 4 °C released proteins were cleared from the beads by centrifugation at 1,000 rpm for 5 min at 4 °C. GST pull-down protein fractions were used for analysis by mass spectrometry.

2022-01-17 | PXD030058 | Pride

Expression data from HMC-1 mast cells

Project description:We demonstrate that the G protein Gi3 is the cellular target of the adenosine A3 receptor (A3R). By using a cell permeable peptide comprising the C-terminal end of Gαi3 fused to an importation sequence (ALL1) as a selective inhibitor of Gi3 signaling, we show that by coupling to Gi3, the A3R stimulates multiple signaling pathways in human mast cells, leading to upregulation of cytokines, chemokines and growth factors.Following contact with activated T cell membranes, endogenous adenosine binds to and activates the A3R, resulting in Gi3-mediated signaling. Specifically, the majority of ERK1/2 signaling initiated by contact with activated T cell membranes, is mediated by Gi3, giving rise to ALL1-inhibitable cellular responses. These results unveil the physiological GPCR that couples to Gi3 and establish the important role played by this G-protein in inflammatory conditions that involve adenosine-activated mast cells. We used microarrays to detail the effect of ALL1 on gene expression of HMC-1 cells activated directly by the A3 receptor, or by contact with activated T cell membranes.

2010-03-31 | GSE19888 | GEO

Immune Cell Infiltration Analysis Demonstrates Excessive Mast Cell Activation in Psoriasis.

Project description: Not available

| S-EPMC8650163 | biostudies-literature

Sequence variation within similar cis-elements promotes context-specific functions of two Drosophila GA-binding transcription factors (protein binding microarray data set)

Project description:To more specifically define the sequence content that favors direct binding of GAF, a genomic-context protein binding microarray (gcPBM) was used to detect GST-tagged GAF binding to double stranded DNA probes using a fluorescently conjugated anti-GST secondary antibody

2018-02-15 | GSE110653 | GEO

Identification of SOX2-bound mRNA

Project description:In vitro pull down assay was performed using recombinant GST and GST fusion SOX2 protein to precipitate total RNA from urothelial carcinoma cell line BFTC905 Unbound RNA from both reactions and matrix-associated RNA from SOX2 pull down reaction was extracted using TRIZOL reagent

2011-10-26 | GSE33207 | GEO

Murine liver: Sperm vs. seminal plasma influences

Project description:Transcriptional profiling of adult mouse liver tissue comparing offspring derived from sperm and seminal plasma of normal protein diet fed males (controls, NN), sperm and seminal plasma from males fed a low protein diet fed males (LL), sperm from normal protein fed males and seminal plasma from low protein fed males (NL) or sperm from low protein diet fed males and seminal plasma from normal protein diet males (NL). The first letter denotes the diet of the sperm donor and the second letter the diet of the seminal plasma donor. Three-condition experiment: NN vs. LL, NN vs. NL, NN vs. LN. Adult offspring liver tissue. Biological replicates: 7 control (NN), 9 LL, 7 NL and 7 LN. One replicate per array chip.

2022-09-20 | GSE211520 | GEO

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