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on-line reverse phase nano high-performance liquid chromatography  (Eksigent Technologies LLC)

 
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    Eksigent Technologies LLC on-line reverse phase nano high-performance liquid chromatography
    On Line Reverse Phase Nano High Performance Liquid Chromatography, supplied by Eksigent Technologies LLC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/phase+separation+buffer/nano+liquid+chromatography+system/us10048266-166-9-22
    Average 90 stars, based on 1 article reviews
    on-line reverse phase nano high-performance liquid chromatography - by Bioz Stars, 2026-10
    90/100 stars

    Images

    Related Articles

    Chromatography:

    Article Title: Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Article Title: Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination.
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLCMS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Mass Spectrometry:

    Article Title: Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Article Title: Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination.
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLCMS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Data-dependent acquisition:

    Article Title: Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Article Title: Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination.
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLCMS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Liquid Chromatography:

    Article Title: Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    Article Title: Vitreous Proteomics Provides New Insights into Antivascular Endothelial Growth Factor Therapy for Pathologic Myopia Choroid Neovascularization.
    Article Snippet: This study aimed to investigate the protein expression profile of vitreous humor (VH) from pathologic myopic retinoschisis (PMRS) patients with or without intravitreal antivascular endothelial growth factor (anti-VEGF) therapy.. VH samples from PMRS patients were subjected to proteomic analysis.. Clinical data, including visual acuity, refractive error, and axial length, were recorded, and the fundus optical coherence tomography was performed.

    Article Title: Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination.
    Article Snippet: .. The resulting peptide mixture was then analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLCMS/MS) using data-dependent acquisition on cHiPLC nano liquid chromatography system (Eksigent) and TripleTOF 5600 mass spectrometer (ABSCIEX). ..

    other:

    Article Title: Screening and Evaluation of Novel α-Glucosidase Inhibitory Peptides from Ginkgo biloba Seed Cake Based on Molecular Docking Combined with Molecular Dynamics Simulation.
    Article Snippet: 283 NEGRRVFDGAVK 11S-globulin 1346.708 449.909 3 0.171 284 GFDTDTLAEAMGISQ Ginnacin, 11S-globulin 1597.770 799.892 2 0.171 285 ETLNPNALSLPRYTN Ginnacin, 11S-globulin 1701.870 851.942 2 0.171 286 GEGRLGVV Ginnacin, 11S-globulin 785.441 393.728 2 0.171 287 MGISQDTAR Ginnacin, 11S-globulin 977.458 489.736 2 0.171 288 IVAIADTSNDQNQLD Ginnacin 1615.772 808.893 2 0.061 289 IADTSNHQNQLDQ 11S-globulin 1483.659 742.837 2 0.060 290 IVAIADTSNDQNQ Ginnacin 1386.664 694.339 2 0.060 291 DTSNDQNQLDQ Ginnacin 1276.521 639.268 2 0.060 292 TSNDQNQLDQT Ginnacin 1262.543 632.279 2 0.059 Table S2 The docking scores and toxicity of the peptides Number Sequence Docking score Toxicity 1 FAPSW -7.46 Non-Toxin 2 MPGPP -7.09 Non-Toxin Table S3 The information of the instrument, parameters and the gradient elution conditions Item Information Instrument AB Eksigent EkspertTM nano liquid chromatography (LC 425)- tandem Sciex Triple TOFTM 5600 plus mass spectrometry (AB Sciex, Foster City, CA, USA) Chromatographic column 3C18-CL-120 chromatographic column (0.3×150 mm, 3 μm, Eksigent, USA).

    Article Title: Identification of CSPG4 as a Biomarker and Therapeutic Target for Infantile Post-Hemorrhagic Hydrocephalus via Multi-Omics Analysis.
    Article Snippet: Then the peptides were injected into an Eksigent nano High Performance Liquid Chromatography (HPLC) system.

    Article Title: Protein profiling identified mitochondrial dysfunction and synaptic abnormalities after dexamethasone intervention in rats with traumatic brain injury.
    Article Snippet: The digested products were mixed and analyzed by liquid chromatography (Eksigent, Framingham, MA, USA) (strong cation exchange column 4.6 × 250 mm, 5 μm-C18, 100 Å).

    Article Title: CAPG enhances breast cancer metastasis by competing with PRMT5 to modulate STC-1 transcription
    Article Snippet: The trypsin-digested peptides were fractionated and separated using nanoscale high-performance liquid chromatography (nano-HPLC) (Eksigent Technologies) on a secondary reverse-phase (RP) analytical column.

    Article Title: Proteomic Study Between Interstitial Channels Along Meridians and Adjacent Areas in Mini-Pigs.
    Article Snippet: Using a nano-high-performance liquid chromatography (HPLC) system (Eksigent 400 nano HPLC) (Eksigent, AB Sciex, Dublin, CA, USA) coupled to a tandem time-of-flight mass spectrometer (5600 + TripleTOF) (AB Sciex, Dublin, CA, USA), data-dependent acquisition (DDA) mode was employed to optimize the chromatographic and mass spectrometric system parameters by collecting data on the mixed samples.



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    a DIC images of FLAG-mIRS-1 LLPS at a series of protein concentrations (5–50 μM) (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 150 mM NaCl). The proteins were incubated with phase <t>separation</t> buffer at room temperature for 40 min. Scale bar, 20 µm. b DIC images of FLAG-mIRS-1 LLPS in the presence of molecular crowding (2% w/v PEG-8000) (right). No phase separation was observed without crowding agent (left). The proteins were incubated with phase separation buffer at room temperature for 5 min. Scale bar, 20 µm. c LLPS of mIRS-1 at 37 °C or 4 °C for 10 min. Scale bar, 20 µm. d DIC images of FLAG-mIRS-1 LLPS under different salinity, as indicated. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. e DIC images of FLAG-mIRS-1 LLPS with the addition of 1,6-hexanediol at indicated concentration. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. f Phase diagrams of GFP-mIRS-1 with concentrations ranging from 0.1-1.6 μM in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM). Blue dots indicate no phase separation. Red dots indicate phase separation. The LLPS ability of mIRS-1 under different conditions was color-coded on the basis of droplet turbidity as measured at OD600 when the proteins had been incubated with phase separation buffer at 37 °C for 120 min. g Quantification result of endogenous IRS-1 protein concentrations in C2C12 cells based on immunoblot densitometry analysis performed on cell lysates and purified FLAG-mIRS-1 protein.
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    a DIC images of FLAG-mIRS-1 LLPS at a series of protein concentrations (5–50 μM) (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 150 mM NaCl). The proteins were incubated with phase <t>separation</t> buffer at room temperature for 40 min. Scale bar, 20 µm. b DIC images of FLAG-mIRS-1 LLPS in the presence of molecular crowding (2% w/v PEG-8000) (right). No phase separation was observed without crowding agent (left). The proteins were incubated with phase separation buffer at room temperature for 5 min. Scale bar, 20 µm. c LLPS of mIRS-1 at 37 °C or 4 °C for 10 min. Scale bar, 20 µm. d DIC images of FLAG-mIRS-1 LLPS under different salinity, as indicated. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. e DIC images of FLAG-mIRS-1 LLPS with the addition of 1,6-hexanediol at indicated concentration. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. f Phase diagrams of GFP-mIRS-1 with concentrations ranging from 0.1-1.6 μM in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM). Blue dots indicate no phase separation. Red dots indicate phase separation. The LLPS ability of mIRS-1 under different conditions was color-coded on the basis of droplet turbidity as measured at OD600 when the proteins had been incubated with phase separation buffer at 37 °C for 120 min. g Quantification result of endogenous IRS-1 protein concentrations in C2C12 cells based on immunoblot densitometry analysis performed on cell lysates and purified FLAG-mIRS-1 protein.
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    a DIC images of FLAG-mIRS-1 LLPS at a series of protein concentrations (5–50 μM) (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 150 mM NaCl). The proteins were incubated with phase <t>separation</t> buffer at room temperature for 40 min. Scale bar, 20 µm. b DIC images of FLAG-mIRS-1 LLPS in the presence of molecular crowding (2% w/v PEG-8000) (right). No phase separation was observed without crowding agent (left). The proteins were incubated with phase separation buffer at room temperature for 5 min. Scale bar, 20 µm. c LLPS of mIRS-1 at 37 °C or 4 °C for 10 min. Scale bar, 20 µm. d DIC images of FLAG-mIRS-1 LLPS under different salinity, as indicated. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. e DIC images of FLAG-mIRS-1 LLPS with the addition of 1,6-hexanediol at indicated concentration. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. f Phase diagrams of GFP-mIRS-1 with concentrations ranging from 0.1-1.6 μM in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM). Blue dots indicate no phase separation. Red dots indicate phase separation. The LLPS ability of mIRS-1 under different conditions was color-coded on the basis of droplet turbidity as measured at OD600 when the proteins had been incubated with phase separation buffer at 37 °C for 120 min. g Quantification result of endogenous IRS-1 protein concentrations in C2C12 cells based on immunoblot densitometry analysis performed on cell lysates and purified FLAG-mIRS-1 protein.
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    Image Search Results


    a DIC images of FLAG-mIRS-1 LLPS at a series of protein concentrations (5–50 μM) (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 150 mM NaCl). The proteins were incubated with phase separation buffer at room temperature for 40 min. Scale bar, 20 µm. b DIC images of FLAG-mIRS-1 LLPS in the presence of molecular crowding (2% w/v PEG-8000) (right). No phase separation was observed without crowding agent (left). The proteins were incubated with phase separation buffer at room temperature for 5 min. Scale bar, 20 µm. c LLPS of mIRS-1 at 37 °C or 4 °C for 10 min. Scale bar, 20 µm. d DIC images of FLAG-mIRS-1 LLPS under different salinity, as indicated. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. e DIC images of FLAG-mIRS-1 LLPS with the addition of 1,6-hexanediol at indicated concentration. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. f Phase diagrams of GFP-mIRS-1 with concentrations ranging from 0.1-1.6 μM in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM). Blue dots indicate no phase separation. Red dots indicate phase separation. The LLPS ability of mIRS-1 under different conditions was color-coded on the basis of droplet turbidity as measured at OD600 when the proteins had been incubated with phase separation buffer at 37 °C for 120 min. g Quantification result of endogenous IRS-1 protein concentrations in C2C12 cells based on immunoblot densitometry analysis performed on cell lysates and purified FLAG-mIRS-1 protein.

    Journal: Cell Discovery

    Article Title: Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes

    doi: 10.1038/s41421-022-00426-x

    Figure Lengend Snippet: a DIC images of FLAG-mIRS-1 LLPS at a series of protein concentrations (5–50 μM) (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 150 mM NaCl). The proteins were incubated with phase separation buffer at room temperature for 40 min. Scale bar, 20 µm. b DIC images of FLAG-mIRS-1 LLPS in the presence of molecular crowding (2% w/v PEG-8000) (right). No phase separation was observed without crowding agent (left). The proteins were incubated with phase separation buffer at room temperature for 5 min. Scale bar, 20 µm. c LLPS of mIRS-1 at 37 °C or 4 °C for 10 min. Scale bar, 20 µm. d DIC images of FLAG-mIRS-1 LLPS under different salinity, as indicated. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. e DIC images of FLAG-mIRS-1 LLPS with the addition of 1,6-hexanediol at indicated concentration. The proteins (10 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. f Phase diagrams of GFP-mIRS-1 with concentrations ranging from 0.1-1.6 μM in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM). Blue dots indicate no phase separation. Red dots indicate phase separation. The LLPS ability of mIRS-1 under different conditions was color-coded on the basis of droplet turbidity as measured at OD600 when the proteins had been incubated with phase separation buffer at 37 °C for 120 min. g Quantification result of endogenous IRS-1 protein concentrations in C2C12 cells based on immunoblot densitometry analysis performed on cell lysates and purified FLAG-mIRS-1 protein.

    Article Snippet: The purified proteins were added to phase separation Buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 2% PEG-8000 (Sangon biotech, A100159)).

    Techniques: Incubation, Concentration Assay, Western Blot, Purification

    a Schematic diagram of IDR of mIRS-1 and its truncation mutants. b FLAG-tagged mIRS-1 mutant constructs as shown in ( a ) were co-transfected with GFP-mIRS-1 IDR (301–1233) into 293 T cells for immunoprecipitation analysis. c Schematic diagram of mIRS-1 and its deletion mutants. d FLAG-tagged and GFP-tagged mIRS-1 or mIRS-1 ΔSAR mutants were co-transfected into 293 T cells for immunoprecipitation analysis. Data in the bar graphs represent the means ± SEM values of the ratios of densities for three independent experiments. *** p < 0.001. e FLAG-tagged mIRS-1 mutant constructs were co-transfected with GFP-mIRS-1 301–600 into 293 T cells for immunoprecipitation analysis. f Representative confocal images of GFP-tagged mIRS-1, mIRS-1 ΔSAR mutant, and mIRS-1-TDP-43 mutant in C2C12 cells. Quantification results of GFP-mIRS-1 and mutant puncta are shown as violin plots ( n = 80). **** p < 0.0001. ns: not significant. g Confocal images and quantification of GFP-mIRS-1-TDP-43 mutant fluorescence recovery after photobleaching. Scale bar, 1 µm. h Time-lapse imaging showing fusion of two GFP-mIRS-1-TDP-43 droplets in cells. Scale bar, 1 µm. i Immunoblot analysis of the indicated proteins in C2C12-IRS-1 KO cell lines stably expressing GFP-mIRS-1 (mIRS-1 WT, mIRS-1 ΔSAR, mIRS-1 Δ600–800, mIRS-1 Δ800–1000, and mIRS-1 Δ1001–1233). j Confocal images of GFP-tagged mIRS-1 wildtype and mutants in C2C12 cell lines as indicated in ( i ). Scale bar, 10 µm. Quantification results of GFP-mIRS-1 or mutant puncta are shown as violin plots. **** p < 0.0001. ns not significant. k DIC images of FLAG-mIRS-1 and mIRS-1 ΔSAR LLPS. The proteins (1 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm.

    Journal: Cell Discovery

    Article Title: Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes

    doi: 10.1038/s41421-022-00426-x

    Figure Lengend Snippet: a Schematic diagram of IDR of mIRS-1 and its truncation mutants. b FLAG-tagged mIRS-1 mutant constructs as shown in ( a ) were co-transfected with GFP-mIRS-1 IDR (301–1233) into 293 T cells for immunoprecipitation analysis. c Schematic diagram of mIRS-1 and its deletion mutants. d FLAG-tagged and GFP-tagged mIRS-1 or mIRS-1 ΔSAR mutants were co-transfected into 293 T cells for immunoprecipitation analysis. Data in the bar graphs represent the means ± SEM values of the ratios of densities for three independent experiments. *** p < 0.001. e FLAG-tagged mIRS-1 mutant constructs were co-transfected with GFP-mIRS-1 301–600 into 293 T cells for immunoprecipitation analysis. f Representative confocal images of GFP-tagged mIRS-1, mIRS-1 ΔSAR mutant, and mIRS-1-TDP-43 mutant in C2C12 cells. Quantification results of GFP-mIRS-1 and mutant puncta are shown as violin plots ( n = 80). **** p < 0.0001. ns: not significant. g Confocal images and quantification of GFP-mIRS-1-TDP-43 mutant fluorescence recovery after photobleaching. Scale bar, 1 µm. h Time-lapse imaging showing fusion of two GFP-mIRS-1-TDP-43 droplets in cells. Scale bar, 1 µm. i Immunoblot analysis of the indicated proteins in C2C12-IRS-1 KO cell lines stably expressing GFP-mIRS-1 (mIRS-1 WT, mIRS-1 ΔSAR, mIRS-1 Δ600–800, mIRS-1 Δ800–1000, and mIRS-1 Δ1001–1233). j Confocal images of GFP-tagged mIRS-1 wildtype and mutants in C2C12 cell lines as indicated in ( i ). Scale bar, 10 µm. Quantification results of GFP-mIRS-1 or mutant puncta are shown as violin plots. **** p < 0.0001. ns not significant. k DIC images of FLAG-mIRS-1 and mIRS-1 ΔSAR LLPS. The proteins (1 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm.

    Article Snippet: The purified proteins were added to phase separation Buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 2% PEG-8000 (Sangon biotech, A100159)).

    Techniques: Mutagenesis, Construct, Transfection, Immunoprecipitation, Fluorescence, Imaging, Western Blot, Stable Transfection, Expressing, Incubation

    a Confocal images of GFP-mIRS-1 foci in C2C12-IRS-1 KO/GFP-mIRS-1 cells treated with control or with IGF-1-conditioned (100 ng/mL) medium for 2.5 min. Scale bar, 10 µm. Quantitative analysis of number of mIRS-1 puncta is shown with results presented as violin plots. *** p < 0.001. b FLAG-tagged and GFP-tagged mIRS-1 were co-transfected into 293 T cells. Cells were serum starved for 16 h followed by IGF-1 stimulation and coimmunoprecipitation analysis. c Immunofluorescence staining of endogenous p-IRS-1 (Y608) antibody in C2C12-IRS-1 KO/GFP-mIRS-1 cells treated for 5 min with control medium or IGF-1-conditioned medium. Scale bar, 5 µm. Line scan showing the related intensity profiles of mIRS-1 with p-IRS-1 (Y608). The puncta diameter was quantified ( n = 69). Data in the bar graphs represent the means ± SEM. d Immunoblot analysis of Y608 residue tyrosine phosphorylation of FLAG-mIRS-1 purified from starved or insulin-stimulated (15 min) 293 T cells. Quantification result is shown as means ± SEM. ** p < 0.001. e DIC images of FLAG-mIRS-1 purified from starved or insulin-stimulated (15 min) 293 T cells. The proteins (1 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. Quantification result is shown as means ± SD. ** p < 0.01. f Scheme indicating the location of the nine tyrosine residues of YXXM motifs in mIRS-1. g The nine tyrosine residues of the YXXM motifs in mIRS-1 were replaced by alanine residues. h Immunoblot analysis of the IRS-1 expression levels in C2C12 wildtype, C2C12-IRS-1 KO/GFP-mIRS-1, and C2C12-IRS-1 KO/GFP-mIRS-1 9YA cell lines. i Confocal images of GFP-tagged mIRS-1 or 9YA mutant in C2C12-IRS-1 KO/GFP-mIRS-1 or C2C12-IRS-1 KO/GFP-mIRS-1 9YA cell lines. Scale bar, 10 µm. Quantification results of GFP-mIRS-1 or 9YA puncta are shown and presented as violin plots. **** p < 0.0001. j DIC images of FLAG-mIRS-1 or FLAG-mIRS-1 9YA purified from 293 T cells. The proteins (1 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. k Phase diagrams of mIRS-1 wildtype, ΔSAR, and 9YA mutant proteins purified from insulin-stimulated cell lines in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM).

    Journal: Cell Discovery

    Article Title: Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes

    doi: 10.1038/s41421-022-00426-x

    Figure Lengend Snippet: a Confocal images of GFP-mIRS-1 foci in C2C12-IRS-1 KO/GFP-mIRS-1 cells treated with control or with IGF-1-conditioned (100 ng/mL) medium for 2.5 min. Scale bar, 10 µm. Quantitative analysis of number of mIRS-1 puncta is shown with results presented as violin plots. *** p < 0.001. b FLAG-tagged and GFP-tagged mIRS-1 were co-transfected into 293 T cells. Cells were serum starved for 16 h followed by IGF-1 stimulation and coimmunoprecipitation analysis. c Immunofluorescence staining of endogenous p-IRS-1 (Y608) antibody in C2C12-IRS-1 KO/GFP-mIRS-1 cells treated for 5 min with control medium or IGF-1-conditioned medium. Scale bar, 5 µm. Line scan showing the related intensity profiles of mIRS-1 with p-IRS-1 (Y608). The puncta diameter was quantified ( n = 69). Data in the bar graphs represent the means ± SEM. d Immunoblot analysis of Y608 residue tyrosine phosphorylation of FLAG-mIRS-1 purified from starved or insulin-stimulated (15 min) 293 T cells. Quantification result is shown as means ± SEM. ** p < 0.001. e DIC images of FLAG-mIRS-1 purified from starved or insulin-stimulated (15 min) 293 T cells. The proteins (1 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. Quantification result is shown as means ± SD. ** p < 0.01. f Scheme indicating the location of the nine tyrosine residues of YXXM motifs in mIRS-1. g The nine tyrosine residues of the YXXM motifs in mIRS-1 were replaced by alanine residues. h Immunoblot analysis of the IRS-1 expression levels in C2C12 wildtype, C2C12-IRS-1 KO/GFP-mIRS-1, and C2C12-IRS-1 KO/GFP-mIRS-1 9YA cell lines. i Confocal images of GFP-tagged mIRS-1 or 9YA mutant in C2C12-IRS-1 KO/GFP-mIRS-1 or C2C12-IRS-1 KO/GFP-mIRS-1 9YA cell lines. Scale bar, 10 µm. Quantification results of GFP-mIRS-1 or 9YA puncta are shown and presented as violin plots. **** p < 0.0001. j DIC images of FLAG-mIRS-1 or FLAG-mIRS-1 9YA purified from 293 T cells. The proteins (1 μM) were incubated with phase separation buffer at room temperature for 20 min. Scale bar, 20 µm. k Phase diagrams of mIRS-1 wildtype, ΔSAR, and 9YA mutant proteins purified from insulin-stimulated cell lines in 50 mM Tris (pH 7.5), 2% (w/v) PEG-8000, and sodium chloride (ranging from 50–1200 mM).

    Article Snippet: The purified proteins were added to phase separation Buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 2% PEG-8000 (Sangon biotech, A100159)).

    Techniques: Control, Transfection, Immunofluorescence, Staining, Western Blot, Residue, Phospho-proteomics, Purification, Incubation, Expressing, Mutagenesis

    a DIC images of LLPS of FLAG-hIRS-1 and FLAG-hIRS-1 G972 mutants at a series of protein concentrations (5 or 10 μM). The proteins were incubated with phase separation buffer at room temperature for 10 min. Scale bar, 20 µm . b Immunoblot analysis of the IRS-1 expression levels in C2C12 wildtype, C2C12-IRS-1 KO/GFP-hIRS-1, and C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines. c Confocal images of GFP-tagged hIRS-1 or G972R mutant in C2C12-IRS-1 KO/GFP-hIRS-1 or C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines. Scale bar, 10 µm. Quantification results of GFP-hIRS-1 or G972R puncta are shown as violin plots. * p < 0.05. d FLAG-tagged hIRS-1 or hIRS-1 G972R mutants were co-transfected with GFP-hIRS-1 into 293 T cells for immunoprecipitation analysis. Data in the bar graphs represent the mean ± SEM values of the ratios of densities for three independent experiments. *** p < 0.001. e FLAG-tagged mIRS-1 801–1000 or mIRS-1 801–1000 G965R mutant was co-transfected with GFP-mIRS-1-301-600 into 293 T cells for immunoprecipitation analysis. Data in the bar graphs represent the mean ± SEM values of the ratios of densities for three independent experiments. **** p < 0.0001. f Immunoblot analysis of total and phosphorylated AKT and ERK levels in C2C12 wildtype, C2C12-IRS-1 KO, C2C12-IRS-1 KO/GFP-hIRS-1, or C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines treated with or without IGF-1 conditional medium for 2.5 min. Data in the bar graphs represent the means ± SEM values of the ratios of densities for three independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001. ns: not significant. g GFP-tagged hIRS-1 wildtype and G972R mutant were immunoprecipitated in IGF-1-stimulated or control C2C12-IRS-1 KO/GFP-hIRS-1, or C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines and then subjected to Western blot with p85 or Grb2 antibodies. Data in the bar graphs represent the mean ± SEM values of the ratios of densities for three independent experiments. * p < 0.05. *** p < 0.001. h Confocal images of endogenous p85 and GFP-hIRS-1 or G972R mutants in the indicated cell lines. Scale bar, 10 µm. Line scan showing the related intensity profiles of hIRS-1 with p85. The GFP-hIRS-1 or mutant puncta co-localized with p85 were quantified ( n = 33). Data in the bar graphs represent the means ± SEM. **** p < 0.0001.

    Journal: Cell Discovery

    Article Title: Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes

    doi: 10.1038/s41421-022-00426-x

    Figure Lengend Snippet: a DIC images of LLPS of FLAG-hIRS-1 and FLAG-hIRS-1 G972 mutants at a series of protein concentrations (5 or 10 μM). The proteins were incubated with phase separation buffer at room temperature for 10 min. Scale bar, 20 µm . b Immunoblot analysis of the IRS-1 expression levels in C2C12 wildtype, C2C12-IRS-1 KO/GFP-hIRS-1, and C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines. c Confocal images of GFP-tagged hIRS-1 or G972R mutant in C2C12-IRS-1 KO/GFP-hIRS-1 or C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines. Scale bar, 10 µm. Quantification results of GFP-hIRS-1 or G972R puncta are shown as violin plots. * p < 0.05. d FLAG-tagged hIRS-1 or hIRS-1 G972R mutants were co-transfected with GFP-hIRS-1 into 293 T cells for immunoprecipitation analysis. Data in the bar graphs represent the mean ± SEM values of the ratios of densities for three independent experiments. *** p < 0.001. e FLAG-tagged mIRS-1 801–1000 or mIRS-1 801–1000 G965R mutant was co-transfected with GFP-mIRS-1-301-600 into 293 T cells for immunoprecipitation analysis. Data in the bar graphs represent the mean ± SEM values of the ratios of densities for three independent experiments. **** p < 0.0001. f Immunoblot analysis of total and phosphorylated AKT and ERK levels in C2C12 wildtype, C2C12-IRS-1 KO, C2C12-IRS-1 KO/GFP-hIRS-1, or C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines treated with or without IGF-1 conditional medium for 2.5 min. Data in the bar graphs represent the means ± SEM values of the ratios of densities for three independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001. ns: not significant. g GFP-tagged hIRS-1 wildtype and G972R mutant were immunoprecipitated in IGF-1-stimulated or control C2C12-IRS-1 KO/GFP-hIRS-1, or C2C12-IRS-1 KO/GFP-hIRS-1 G972R cell lines and then subjected to Western blot with p85 or Grb2 antibodies. Data in the bar graphs represent the mean ± SEM values of the ratios of densities for three independent experiments. * p < 0.05. *** p < 0.001. h Confocal images of endogenous p85 and GFP-hIRS-1 or G972R mutants in the indicated cell lines. Scale bar, 10 µm. Line scan showing the related intensity profiles of hIRS-1 with p85. The GFP-hIRS-1 or mutant puncta co-localized with p85 were quantified ( n = 33). Data in the bar graphs represent the means ± SEM. **** p < 0.0001.

    Article Snippet: The purified proteins were added to phase separation Buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 2% PEG-8000 (Sangon biotech, A100159)).

    Techniques: Incubation, Western Blot, Expressing, Mutagenesis, Transfection, Immunoprecipitation, Control

    We found that the C-terminus of IRS-1 undergoes phase separation through mediating self-association. Insulin/IGF signaling leads to tyrosine phosphorylation of IRS-1 which promotes the formation of IRS-1 droplets and the recruitment of downstream effectors to form insulin/IGF signalosomes.

    Journal: Cell Discovery

    Article Title: Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes

    doi: 10.1038/s41421-022-00426-x

    Figure Lengend Snippet: We found that the C-terminus of IRS-1 undergoes phase separation through mediating self-association. Insulin/IGF signaling leads to tyrosine phosphorylation of IRS-1 which promotes the formation of IRS-1 droplets and the recruitment of downstream effectors to form insulin/IGF signalosomes.

    Article Snippet: The purified proteins were added to phase separation Buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 2% PEG-8000 (Sangon biotech, A100159)).

    Techniques: Phospho-proteomics