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phase separation buffer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher phase separation buffer
    Phase Separation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/phase+separation+buffer/HEPES/pm37717144-107-5-23
    Average 99 stars, based on 1 article reviews
    phase separation buffer - by Bioz Stars, 2026-09
    99/100 stars

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    Related Articles

    Buffer Exchange:

    Article Title: Crowder titrations enable the quantification of driving forces for macromolecular phase separation
    Article Snippet: .. The buffer exchange into the phase separation buffer (20 mM HEPES pH 7.3, 2 mM DTT) was achieved by using 2 mL Zeba Spin Desalting columns with 7 MWCO (ThermoFisher). ..

    Article Title: Crowder titrations enable the quantification of driving forces for macromolecular phase separation.
    Article Snippet: .. The buffer exchange into the phase separation buffer (20 mM HEPES [pH 7.3], 2 mMDTT) was achieved using 2 mL Zeba SpinDesalting columnswith 7MWCO(ThermoFisher Scientific, Waltham,MA). ..

    Purification:

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress.
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Ubiquitin Proteomics:

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress.
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Incubation:

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress.
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Microscopy:

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress.
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..

    Article Title: Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress
    Article Snippet: .. Purified 5 μM mCherry-p62 protein and 1.5 μM 8 × ubiquitin protein were incubated in phase separation buffer (40 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT) on the chambered cover glass (Thermo Scientific, 155411) which was previously coated with 1 mg/ml BSA for the indicated time at RT, then the reaction was imaged by confocal microscope. ..



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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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    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