rhoa gtp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rhoa gtp
    Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on <t>RhoA-GTP,</t> RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.
    Rhoa Gtp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhoa gtp - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm"

    Article Title: Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2022/9964689

    Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.
    Figure Legend Snippet: Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.

    Techniques Used: Activation Assay, Western Blot, Derivative Assay, Expressing, Activity Assay, Fluorescence, shRNA

    rhoa gtp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rhoa gtp
    Rhoa Gtp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhoa gtp/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    gtp rhoa  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gtp rhoa
    Gtp Rhoa, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    gtp rhoa assay aml12 cells  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gtp rhoa assay aml12 cells
    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of <t>AML12</t> cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.
    Gtp Rhoa Assay Aml12 Cells, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    gtp rhoa assay aml12 cells - by Bioz Stars, 2024-06
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    1) Product Images from "Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis"

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2020.03.010

    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.
    Figure Legend Snippet: (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.

    Techniques Used: Activity Assay, Incubation

    (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.
    Figure Legend Snippet: (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.

    Techniques Used: Fluorescence, Transduction, Incubation

    (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).
    Figure Legend Snippet: (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).

    Techniques Used: Western Blot, Transfection, Incubation

    (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).
    Figure Legend Snippet: (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).

    Techniques Used: Activity Assay, Incubation

    gtp rhoa assay aml12 cells  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gtp rhoa assay aml12 cells
    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of <t>AML12</t> cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.
    Gtp Rhoa Assay Aml12 Cells, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gtp rhoa assay aml12 cells/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gtp rhoa assay aml12 cells - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis"

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2020.03.010

    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.
    Figure Legend Snippet: (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.

    Techniques Used: Activity Assay, Incubation

    (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.
    Figure Legend Snippet: (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.

    Techniques Used: Fluorescence, Transduction, Incubation

    (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).
    Figure Legend Snippet: (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).

    Techniques Used: Western Blot, Transfection, Incubation

    (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).
    Figure Legend Snippet: (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).

    Techniques Used: Activity Assay, Incubation

    rhoa gtp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rhoa gtp
    Rhoa Gtp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhoa gtp/product/Cell Signaling Technology Inc
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    rhoa gtp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rhoa gtp
    aPC–integrin-αvβ3 temporally regulates <t>RhoA</t> activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-β3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose concentration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG+PBS to HG+aPC. Scale bar, 20 μm. (B) Representative immunoblot images showing levels of <t>RhoA-GTP</t> (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and dot blot summarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 20 nM). (C) Representative integrin-β3 immunoblot images (top; IB: β3) of Gα13 immunoprecipitate showing time-dependent interaction of Gα13 with integrin-β3 upon stimulation of human podocytes with aPC. Gα13 (44 kDa) immunoblots were used as loading controls (top; IB: Gα13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Gα13 with integrin-β3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an individual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC+RGDS), in integrin-β3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-β3, aPC-β3KD), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose (HG, 25 mM) induced podocyte migration (as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot, including mean and SEM in (A, B, and E), or as dots representing individual data points from three independent repeat experiments in the line graphs in (C and D). *P<0.05, **P<0.01, ***P<0.005 (A, B, and E). ***P<0.005 (aPC versus time point 0 minute), ### P<0.005 (RGDS + aPC versus time point 0 minute), ++P<0.01, +++P<0.005 (integrin β3KD + aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).
    Rhoa Gtp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhoa gtp/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhoa gtp - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Podocyte Integrin- β 3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy"

    Article Title: Podocyte Integrin- β 3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2019111163

    aPC–integrin-αvβ3 temporally regulates RhoA activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-β3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose concentration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG+PBS to HG+aPC. Scale bar, 20 μm. (B) Representative immunoblot images showing levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and dot blot summarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 20 nM). (C) Representative integrin-β3 immunoblot images (top; IB: β3) of Gα13 immunoprecipitate showing time-dependent interaction of Gα13 with integrin-β3 upon stimulation of human podocytes with aPC. Gα13 (44 kDa) immunoblots were used as loading controls (top; IB: Gα13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Gα13 with integrin-β3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an individual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC+RGDS), in integrin-β3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-β3, aPC-β3KD), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose (HG, 25 mM) induced podocyte migration (as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot, including mean and SEM in (A, B, and E), or as dots representing individual data points from three independent repeat experiments in the line graphs in (C and D). *P<0.05, **P<0.01, ***P<0.005 (A, B, and E). ***P<0.005 (aPC versus time point 0 minute), ### P<0.005 (RGDS + aPC versus time point 0 minute), ++P<0.01, +++P<0.005 (integrin β3KD + aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).
    Figure Legend Snippet: aPC–integrin-αvβ3 temporally regulates RhoA activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-β3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose concentration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG+PBS to HG+aPC. Scale bar, 20 μm. (B) Representative immunoblot images showing levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and dot blot summarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 20 nM). (C) Representative integrin-β3 immunoblot images (top; IB: β3) of Gα13 immunoprecipitate showing time-dependent interaction of Gα13 with integrin-β3 upon stimulation of human podocytes with aPC. Gα13 (44 kDa) immunoblots were used as loading controls (top; IB: Gα13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Gα13 with integrin-β3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an individual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC+RGDS), in integrin-β3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-β3, aPC-β3KD), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose (HG, 25 mM) induced podocyte migration (as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot, including mean and SEM in (A, B, and E), or as dots representing individual data points from three independent repeat experiments in the line graphs in (C and D). *P<0.05, **P<0.01, ***P<0.005 (A, B, and E). ***P<0.005 (aPC versus time point 0 minute), ### P<0.005 (RGDS + aPC versus time point 0 minute), ++P<0.01, +++P<0.005 (integrin β3KD + aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).

    Techniques Used: Activation Assay, Immunofluorescence, Concentration Assay, Staining, Western Blot, Pull Down Assay, Dot Blot, shRNA, Migration, Wound Healing Assay

    Podocyte-specific deletion of integrin-β3 abrogates the cytoprotective effect of aPC in dNP. (A) Representative immunofluorescence images (left) of glomeruli of nondiabetic mice (db/m or control [C]), diabetic mice (db/db or STZ-induced diabetes [DM]), or diabetic mice treated with aPC (db/db+aPC or DM+aPC). The conformation-specific antibody AP5 was used to detect active integrin-β3 (red). Podocytes were identified by nephrin staining (green); yellow reflects the colocalization of AP5 and nephrin. The nuclei were stained with DAPI (blue). Dot plots summarizing the results are shown at the right. Diabetic mice without or with aPC treatment were compared with nondiabetic control mice (db/m or C) and among each other (db/db versus db/db+aPC and DM versus DM+aPC). Scale bar, 10 μm. (B) Dot plot summarizing urine albumin levels (the albumin-creatinine ratio) in control (C) and diabetic (DM) wild-type (WT) mice, β3ΔPod mice and β3ΔPod mice crossed with APChigh mice (β3ΔPod APChigh). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing (D) the data for the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots (left) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and dot plots summarizing the data from the experimental groups (right). (H) Schematic representation of the working model: aPC cannot interact with integrin-αvβ3 in β3ΔPod mice, resulting in unopposed PAR1-RhoA signaling, aggravating podocyte dysfunction and hence promoting dNP in mice with increased aPC levels. The data shown in dot plots represent the mean±SEM of at least ten mice (A), five mice (B and D–F), or four mice (G) per group. *P<0.05, **P<0.01, ***P<0.005. (A and C–E) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, and diabetic mutant mice were compared with diabetic wild-type mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; MFI, mean fluorescent intensity.
    Figure Legend Snippet: Podocyte-specific deletion of integrin-β3 abrogates the cytoprotective effect of aPC in dNP. (A) Representative immunofluorescence images (left) of glomeruli of nondiabetic mice (db/m or control [C]), diabetic mice (db/db or STZ-induced diabetes [DM]), or diabetic mice treated with aPC (db/db+aPC or DM+aPC). The conformation-specific antibody AP5 was used to detect active integrin-β3 (red). Podocytes were identified by nephrin staining (green); yellow reflects the colocalization of AP5 and nephrin. The nuclei were stained with DAPI (blue). Dot plots summarizing the results are shown at the right. Diabetic mice without or with aPC treatment were compared with nondiabetic control mice (db/m or C) and among each other (db/db versus db/db+aPC and DM versus DM+aPC). Scale bar, 10 μm. (B) Dot plot summarizing urine albumin levels (the albumin-creatinine ratio) in control (C) and diabetic (DM) wild-type (WT) mice, β3ΔPod mice and β3ΔPod mice crossed with APChigh mice (β3ΔPod APChigh). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing (D) the data for the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots (left) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and dot plots summarizing the data from the experimental groups (right). (H) Schematic representation of the working model: aPC cannot interact with integrin-αvβ3 in β3ΔPod mice, resulting in unopposed PAR1-RhoA signaling, aggravating podocyte dysfunction and hence promoting dNP in mice with increased aPC levels. The data shown in dot plots represent the mean±SEM of at least ten mice (A), five mice (B and D–F), or four mice (G) per group. *P<0.05, **P<0.01, ***P<0.005. (A and C–E) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, and diabetic mutant mice were compared with diabetic wild-type mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; MFI, mean fluorescent intensity.

    Techniques Used: Immunofluorescence, Staining, Filtration, Transmission Assay, Electron Microscopy, Western Blot, Mutagenesis, Injection

    Integrin-αvβ3 antagonism dose-dependently modulates dNP. (A) Scheme showing the experimental approach and dosing of the integrin-αvβ3 antagonist Cyclo-RGDfv in wild-type mice with STZ-induced persistent hyperglycemia (DM). (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice; diabetic control mice received PBS. (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data. The data shown in the dot plots represent the mean±SEM of at least eight mice (B–F) or five mice (G) per group; each dot represents data from one mouse. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; diabetic mice were compared do nondiabetic mice, and diabetic mice treated with Cyclo-RGDfv were compared with diabetic control mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection.
    Figure Legend Snippet: Integrin-αvβ3 antagonism dose-dependently modulates dNP. (A) Scheme showing the experimental approach and dosing of the integrin-αvβ3 antagonist Cyclo-RGDfv in wild-type mice with STZ-induced persistent hyperglycemia (DM). (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice; diabetic control mice received PBS. (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data. The data shown in the dot plots represent the mean±SEM of at least eight mice (B–F) or five mice (G) per group; each dot represents data from one mouse. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; diabetic mice were compared do nondiabetic mice, and diabetic mice treated with Cyclo-RGDfv were compared with diabetic control mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection.

    Techniques Used: Staining, Filtration, Transmission Assay, Electron Microscopy, Western Blot, Injection

    aPC protects against dNP via its RGD sequence. (A) Experimental design. SCH79797 was used as a PAR1 antagonist in a subgroup of mice to counteract excess PAR1 signaling. (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice. Some diabetic mice received additional SCH79797 treatment (DM+S). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom; transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image only), and (F) tight-slit pore density, reflecting foot process effacement. (G) Representative immunoblots (bottom) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data (top). (H) Schematic representation of the working model: in APChigh mice, aPC activates PAR1 via its proteolytic activity, thus promoting RhoA activation, but at the same time binds to integrin-β3, restricting RhoA activity and resulting in transient RhoA signaling. In contrast, in RGE-APChigh mice, aPC cannot bind to integrin-β3, resulting in sustained RhoA signaling (bottom). SCH79797, a PAR1 antagonist, counteracts the enhanced PAR1 and RhoA signaling in RGE-APChigh mice. The data shown in dot plots represent the mean±SEM of at least six mice per group. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, diabetic mutant mice were compared with diabetic wild-type mice, and diabetic mice treated with SCH79797 were compared with untreated diabetic mice of the same genotype. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; DM+S, diabetic mice treated with the PAR1 antagonist SCH79797.
    Figure Legend Snippet: aPC protects against dNP via its RGD sequence. (A) Experimental design. SCH79797 was used as a PAR1 antagonist in a subgroup of mice to counteract excess PAR1 signaling. (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice. Some diabetic mice received additional SCH79797 treatment (DM+S). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom; transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image only), and (F) tight-slit pore density, reflecting foot process effacement. (G) Representative immunoblots (bottom) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data (top). (H) Schematic representation of the working model: in APChigh mice, aPC activates PAR1 via its proteolytic activity, thus promoting RhoA activation, but at the same time binds to integrin-β3, restricting RhoA activity and resulting in transient RhoA signaling. In contrast, in RGE-APChigh mice, aPC cannot bind to integrin-β3, resulting in sustained RhoA signaling (bottom). SCH79797, a PAR1 antagonist, counteracts the enhanced PAR1 and RhoA signaling in RGE-APChigh mice. The data shown in dot plots represent the mean±SEM of at least six mice per group. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, diabetic mutant mice were compared with diabetic wild-type mice, and diabetic mice treated with SCH79797 were compared with untreated diabetic mice of the same genotype. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; DM+S, diabetic mice treated with the PAR1 antagonist SCH79797.

    Techniques Used: Sequencing, Staining, Filtration, Transmission Assay, Electron Microscopy, Western Blot, Activity Assay, Activation Assay, Mutagenesis, Injection

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    Cell Signaling Technology Inc rhoa gtp
    aPC–integrin-αvβ3 temporally regulates <t>RhoA</t> activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-β3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose concentration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG+PBS to HG+aPC. Scale bar, 20 μm. (B) Representative immunoblot images showing levels of <t>RhoA-GTP</t> (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and dot blot summarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 20 nM). (C) Representative integrin-β3 immunoblot images (top; IB: β3) of Gα13 immunoprecipitate showing time-dependent interaction of Gα13 with integrin-β3 upon stimulation of human podocytes with aPC. Gα13 (44 kDa) immunoblots were used as loading controls (top; IB: Gα13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Gα13 with integrin-β3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an individual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC+RGDS), in integrin-β3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-β3, aPC-β3KD), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose (HG, 25 mM) induced podocyte migration (as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot, including mean and SEM in (A, B, and E), or as dots representing individual data points from three independent repeat experiments in the line graphs in (C and D). *P<0.05, **P<0.01, ***P<0.005 (A, B, and E). ***P<0.005 (aPC versus time point 0 minute), ### P<0.005 (RGDS + aPC versus time point 0 minute), ++P<0.01, +++P<0.005 (integrin β3KD + aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).
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    1) Product Images from "Podocyte Integrin- β 3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy"

    Article Title: Podocyte Integrin- β 3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2019111163

    aPC–integrin-αvβ3 temporally regulates RhoA activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-β3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose concentration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG+PBS to HG+aPC. Scale bar, 20 μm. (B) Representative immunoblot images showing levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and dot blot summarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 20 nM). (C) Representative integrin-β3 immunoblot images (top; IB: β3) of Gα13 immunoprecipitate showing time-dependent interaction of Gα13 with integrin-β3 upon stimulation of human podocytes with aPC. Gα13 (44 kDa) immunoblots were used as loading controls (top; IB: Gα13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Gα13 with integrin-β3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an individual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC+RGDS), in integrin-β3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-β3, aPC-β3KD), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose (HG, 25 mM) induced podocyte migration (as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot, including mean and SEM in (A, B, and E), or as dots representing individual data points from three independent repeat experiments in the line graphs in (C and D). *P<0.05, **P<0.01, ***P<0.005 (A, B, and E). ***P<0.005 (aPC versus time point 0 minute), ### P<0.005 (RGDS + aPC versus time point 0 minute), ++P<0.01, +++P<0.005 (integrin β3KD + aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).
    Figure Legend Snippet: aPC–integrin-αvβ3 temporally regulates RhoA activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-β3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose concentration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG+PBS to HG+aPC. Scale bar, 20 μm. (B) Representative immunoblot images showing levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and dot blot summarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 20 nM). (C) Representative integrin-β3 immunoblot images (top; IB: β3) of Gα13 immunoprecipitate showing time-dependent interaction of Gα13 with integrin-β3 upon stimulation of human podocytes with aPC. Gα13 (44 kDa) immunoblots were used as loading controls (top; IB: Gα13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Gα13 with integrin-β3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an individual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC+RGDS), in integrin-β3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-β3, aPC-β3KD), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose (HG, 25 mM) induced podocyte migration (as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot, including mean and SEM in (A, B, and E), or as dots representing individual data points from three independent repeat experiments in the line graphs in (C and D). *P<0.05, **P<0.01, ***P<0.005 (A, B, and E). ***P<0.005 (aPC versus time point 0 minute), ### P<0.005 (RGDS + aPC versus time point 0 minute), ++P<0.01, +++P<0.005 (integrin β3KD + aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).

    Techniques Used: Activation Assay, Immunofluorescence, Concentration Assay, Staining, Western Blot, Pull Down Assay, Dot Blot, shRNA, Migration, Wound Healing Assay

    Podocyte-specific deletion of integrin-β3 abrogates the cytoprotective effect of aPC in dNP. (A) Representative immunofluorescence images (left) of glomeruli of nondiabetic mice (db/m or control [C]), diabetic mice (db/db or STZ-induced diabetes [DM]), or diabetic mice treated with aPC (db/db+aPC or DM+aPC). The conformation-specific antibody AP5 was used to detect active integrin-β3 (red). Podocytes were identified by nephrin staining (green); yellow reflects the colocalization of AP5 and nephrin. The nuclei were stained with DAPI (blue). Dot plots summarizing the results are shown at the right. Diabetic mice without or with aPC treatment were compared with nondiabetic control mice (db/m or C) and among each other (db/db versus db/db+aPC and DM versus DM+aPC). Scale bar, 10 μm. (B) Dot plot summarizing urine albumin levels (the albumin-creatinine ratio) in control (C) and diabetic (DM) wild-type (WT) mice, β3ΔPod mice and β3ΔPod mice crossed with APChigh mice (β3ΔPod APChigh). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing (D) the data for the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots (left) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and dot plots summarizing the data from the experimental groups (right). (H) Schematic representation of the working model: aPC cannot interact with integrin-αvβ3 in β3ΔPod mice, resulting in unopposed PAR1-RhoA signaling, aggravating podocyte dysfunction and hence promoting dNP in mice with increased aPC levels. The data shown in dot plots represent the mean±SEM of at least ten mice (A), five mice (B and D–F), or four mice (G) per group. *P<0.05, **P<0.01, ***P<0.005. (A and C–E) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, and diabetic mutant mice were compared with diabetic wild-type mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; MFI, mean fluorescent intensity.
    Figure Legend Snippet: Podocyte-specific deletion of integrin-β3 abrogates the cytoprotective effect of aPC in dNP. (A) Representative immunofluorescence images (left) of glomeruli of nondiabetic mice (db/m or control [C]), diabetic mice (db/db or STZ-induced diabetes [DM]), or diabetic mice treated with aPC (db/db+aPC or DM+aPC). The conformation-specific antibody AP5 was used to detect active integrin-β3 (red). Podocytes were identified by nephrin staining (green); yellow reflects the colocalization of AP5 and nephrin. The nuclei were stained with DAPI (blue). Dot plots summarizing the results are shown at the right. Diabetic mice without or with aPC treatment were compared with nondiabetic control mice (db/m or C) and among each other (db/db versus db/db+aPC and DM versus DM+aPC). Scale bar, 10 μm. (B) Dot plot summarizing urine albumin levels (the albumin-creatinine ratio) in control (C) and diabetic (DM) wild-type (WT) mice, β3ΔPod mice and β3ΔPod mice crossed with APChigh mice (β3ΔPod APChigh). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing (D) the data for the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots (left) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and dot plots summarizing the data from the experimental groups (right). (H) Schematic representation of the working model: aPC cannot interact with integrin-αvβ3 in β3ΔPod mice, resulting in unopposed PAR1-RhoA signaling, aggravating podocyte dysfunction and hence promoting dNP in mice with increased aPC levels. The data shown in dot plots represent the mean±SEM of at least ten mice (A), five mice (B and D–F), or four mice (G) per group. *P<0.05, **P<0.01, ***P<0.005. (A and C–E) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, and diabetic mutant mice were compared with diabetic wild-type mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; MFI, mean fluorescent intensity.

    Techniques Used: Immunofluorescence, Staining, Filtration, Transmission Assay, Electron Microscopy, Western Blot, Mutagenesis, Injection

    Integrin-αvβ3 antagonism dose-dependently modulates dNP. (A) Scheme showing the experimental approach and dosing of the integrin-αvβ3 antagonist Cyclo-RGDfv in wild-type mice with STZ-induced persistent hyperglycemia (DM). (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice; diabetic control mice received PBS. (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data. The data shown in the dot plots represent the mean±SEM of at least eight mice (B–F) or five mice (G) per group; each dot represents data from one mouse. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; diabetic mice were compared do nondiabetic mice, and diabetic mice treated with Cyclo-RGDfv were compared with diabetic control mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection.
    Figure Legend Snippet: Integrin-αvβ3 antagonism dose-dependently modulates dNP. (A) Scheme showing the experimental approach and dosing of the integrin-αvβ3 antagonist Cyclo-RGDfv in wild-type mice with STZ-induced persistent hyperglycemia (DM). (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice; diabetic control mice received PBS. (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data. The data shown in the dot plots represent the mean±SEM of at least eight mice (B–F) or five mice (G) per group; each dot represents data from one mouse. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; diabetic mice were compared do nondiabetic mice, and diabetic mice treated with Cyclo-RGDfv were compared with diabetic control mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection.

    Techniques Used: Staining, Filtration, Transmission Assay, Electron Microscopy, Western Blot, Injection

    aPC protects against dNP via its RGD sequence. (A) Experimental design. SCH79797 was used as a PAR1 antagonist in a subgroup of mice to counteract excess PAR1 signaling. (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice. Some diabetic mice received additional SCH79797 treatment (DM+S). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom; transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image only), and (F) tight-slit pore density, reflecting foot process effacement. (G) Representative immunoblots (bottom) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data (top). (H) Schematic representation of the working model: in APChigh mice, aPC activates PAR1 via its proteolytic activity, thus promoting RhoA activation, but at the same time binds to integrin-β3, restricting RhoA activity and resulting in transient RhoA signaling. In contrast, in RGE-APChigh mice, aPC cannot bind to integrin-β3, resulting in sustained RhoA signaling (bottom). SCH79797, a PAR1 antagonist, counteracts the enhanced PAR1 and RhoA signaling in RGE-APChigh mice. The data shown in dot plots represent the mean±SEM of at least six mice per group. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, diabetic mutant mice were compared with diabetic wild-type mice, and diabetic mice treated with SCH79797 were compared with untreated diabetic mice of the same genotype. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; DM+S, diabetic mice treated with the PAR1 antagonist SCH79797.
    Figure Legend Snippet: aPC protects against dNP via its RGD sequence. (A) Experimental design. SCH79797 was used as a PAR1 antagonist in a subgroup of mice to counteract excess PAR1 signaling. (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice. Some diabetic mice received additional SCH79797 treatment (DM+S). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 μm) and the glomerular filtration barrier (bottom; transmission electron microscopy; scale bar, 0.2 μm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image only), and (F) tight-slit pore density, reflecting foot process effacement. (G) Representative immunoblots (bottom) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and β-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data (top). (H) Schematic representation of the working model: in APChigh mice, aPC activates PAR1 via its proteolytic activity, thus promoting RhoA activation, but at the same time binds to integrin-β3, restricting RhoA activity and resulting in transient RhoA signaling. In contrast, in RGE-APChigh mice, aPC cannot bind to integrin-β3, resulting in sustained RhoA signaling (bottom). SCH79797, a PAR1 antagonist, counteracts the enhanced PAR1 and RhoA signaling in RGE-APChigh mice. The data shown in dot plots represent the mean±SEM of at least six mice per group. *P<0.05, **P<0.01, ***P<0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, diabetic mutant mice were compared with diabetic wild-type mice, and diabetic mice treated with SCH79797 were compared with untreated diabetic mice of the same genotype. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; DM+S, diabetic mice treated with the PAR1 antagonist SCH79797.

    Techniques Used: Sequencing, Staining, Filtration, Transmission Assay, Electron Microscopy, Western Blot, Activity Assay, Activation Assay, Mutagenesis, Injection

    rhoa gtpor rac1 gtp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rhoa gtp
    Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on <t>RhoA-GTP,</t> RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.
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    Cell Signaling Technology Inc gtp rhoa
    Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on <t>RhoA-GTP,</t> RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.
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    Cell Signaling Technology Inc gtp rhoa assay aml12 cells
    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of <t>AML12</t> cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.
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    Cell Signaling Technology Inc rhoa gtpor rac1 gtp
    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of <t>AML12</t> cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.
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    Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm

    doi: 10.1155/2022/9964689

    Figure Lengend Snippet: Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.

    Article Snippet: Western blot analyses were performed according to standard protocol [ ] using the following antibodies against: α 7-nAChR (ab216485; 1 : 1000), p38 (ab31828; 1 : 1000), p-p38 (phospho T180+Y182) (ab4822; 1 : 1000), IL-6 (ab9324; 1 : 1000), inducible NO synthase (ab3523; 1 : 1000), and GAPDH (ab9484; 1 : 10,000), purchased from Abcam (Abcam plc., Cambridge, UK), and RhoA (#2117; 1 : 1000), RhoA-GTP (#8820; 1 : 1000), ROCK1 (#4035; 1 : 1000), ROCK2 (#9029; 1 : 1000), t-MBS (#2634; 1 : 1000), and p-MBS (#3040; 1 : 1000) from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA) in Supplementary Table .

    Techniques: Activation Assay, Western Blot, Derivative Assay, Expressing, Activity Assay, Fluorescence, shRNA

    (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.

    Journal: Cell metabolism

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    doi: 10.1016/j.cmet.2020.03.010

    Figure Lengend Snippet: (A) RhoA activity (left graph, G-LISA® assay; right graph, GTP-RhoA precipitation using GSTRhotekin-RBD) of AML12 cells incubated for 2 h with vehicle or liposomes, or for 24 h with liposomes and then 2 h with Lipo-Chol. For the G-LISA® assay, n = 4 biological replicates; values shown are means ± SEM; *p < 0.05.

    Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

    Techniques: Activity Assay, Incubation

    (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.

    Journal: Cell metabolism

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    doi: 10.1016/j.cmet.2020.03.010

    Figure Lengend Snippet: (A) Images and mean fluorescence intensity (MFI) per cell of AML12 cells transduced with cyto-GCaMP6f or ER-GCaMP6f and then incubated for 30 min with vehicle or liposomes (n = 4 biological replicates; means ± SEM; *p < 0.05). Scale bar, 100 μm.

    Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

    Techniques: Fluorescence, Transduction, Incubation

    (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).

    Journal: Cell metabolism

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    doi: 10.1016/j.cmet.2020.03.010

    Figure Lengend Snippet: (A) TAZ immunoblot of siScr- or siGnas-transfected AML12 cells that were incubated for 16 h with liposomes and then 8 h with Lipo-Chol).

    Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

    Techniques: Western Blot, Transfection, Incubation

    (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).

    Journal: Cell metabolism

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    doi: 10.1016/j.cmet.2020.03.010

    Figure Lengend Snippet: (A) RhoA activity of AML12 cells that were incubated for 24 h with vehicle or liposomes or for 2 h with liposomes and then 3 h with Lipo-Chol ± 10 μM ALOD4 (n = 6 biological replicates; means ± SEM; ***p < 0.001 vs. both Veh groups).

    Article Snippet: Immunoprecipitation, LATS2 Kinase Assay, and GTP-RhoA Assay AML12 cells from 100-mm culture dishes were collected into 0.4 ml 1x ice-cold cell lysis buffer (#9803S, Cell Signaling) and incubated on ice for 5 min. Then cell lysates were centrifuged at 10,000 x g for 10 min at 4°C.

    Techniques: Activity Assay, Incubation