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biotage phase separator  (BIOTAGE)

 
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    Structured Review

    BIOTAGE biotage phase separator
    Biotage Phase Separator, supplied by BIOTAGE, 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/biotage+phase+separator/us10766903-922-24-24
    Average 90 stars, based on 1 article reviews
    biotage phase separator - by Bioz Stars, 2026-09
    90/100 stars

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    Related Articles

    other:

    Article Title: The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure–Activity Relationships at TRPM PZQ Orthologs
    Article Snippet: The organic layer was passed through a Biotage® phase separator and concentrated to an amorphous solid that was used without further manipulation (111 mg, 94% yield).

    Article Title: The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure–Activity Relationships at TRPM PZQ Orthologs
    Article Snippet: The organic layer was passed through a Biotage® phase separator and concentrated onto Celite®.

    Article Title: The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure–Activity Relationships at TRPM PZQ Orthologs
    Article Snippet: The layers were separated, and the organic layer was passed through a Biotage® phase separator and dry-loaded onto Celite®.

    Article Title: The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure–Activity Relationships at TRPM PZQ Orthologs
    Article Snippet: The organic layer was passed through a Biotage® phase separator and concentrated to an amorphous solid that was used without further manipulation (105 mg, 98% yield).

    Article Title: Organic electroluminescent materials and devices
    Article Snippet: The combined organic layers were dried using a Biotage-phase-separator and solvents removed in vacuo.

    Article Title: The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure–Activity Relationships at TRPM PZQ Orthologs
    Article Snippet: The organic layer was passed through a Biotage® phase separator and dry-loaded onto Celite®.

    Article Title: The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure–Activity Relationships at TRPM PZQ Orthologs
    Article Snippet: The organic layer was passed through a Biotage® phase separator and dry-loaded onto Celite.

    Residue:

    Article Title: Mild Strategy for the Preparation of Alkyl Sulfonyl Fluorides from Alkyl Bromides and Alcohols Using Photoredox Catalysis and Flow Chemistry.
    Article Snippet: .. The residue was resuspended in water (1.5 mL) and extracted with DCM (3×1.5 mL) using Isolute Biotage® phase separator cartridge. .. Combined organic phases were evacuated under reduced pressure to give crude material which was analysed using 19F NMR using trifluorotoluene as internal standard and then purified by flash chromatography (Biotage® Sfär Silica HC D, 5 g column) eluting with EtOAc in heptane (0-60%) to give the title products.



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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