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total rab10  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc total rab10
    Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of <t>Rab10</t> phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).
    Total Rab10, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Doubly Constrained C-terminal of Roc (COR) Domain-Derived Peptides Inhibit Leucine-Rich Repeat Kinase 2 (LRRK2) Dimerization"

    Article Title: Doubly Constrained C-terminal of Roc (COR) Domain-Derived Peptides Inhibit Leucine-Rich Repeat Kinase 2 (LRRK2) Dimerization

    Journal: ACS Chemical Neuroscience

    doi: 10.1021/acschemneuro.3c00259

    Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of Rab10 phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).
    Figure Legend Snippet: Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of Rab10 phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).

    Techniques Used: Activity Assay, Molecular Weight, Purification, Incubation, Cell Surface Biotinylation Assay, Western Blot, Knock-Out, Transfection, Staining



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    a Example of two control and 12 PD LCL lines. Cells were lysed and extracts subjected to quantitative immunoblot analysis with the indicated antibodies and membranes were developed using Odyssey CLx scan Western Blot imaging system. <t>pT73-Rab10</t> and total Rab10, as well as pS935-LRRK2 and total LRRK2 were multiplexed, and the same control line (S001) was run on every gel to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin, pS935/tubulin, pS935/LRRK2, Rab10/tubulin, pT73-Rab10/tubulin and pT73-Rab10/Rab10 as indicated, with no differences observed between PD LCL lines with or without a centrosome splitting phenotype. Bars represent mean ± s.e.m. c Spearman correlation analysis between levels of LRRK2/tubulin and pT73-Rab10/tubulin (top) or pS935/tubulin and pT73-Rab10/tubulin (bottom). A significant association is observed between LRRK2 or S935-LRRK2 levels and pT73-Rab10 levels in PD LCLs. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p -values are indicated for each correlation analysis.
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    Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of <t>Rab10</t> phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).
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    Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of <t>Rab10</t> phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).
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    Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of <t>Rab10</t> phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).
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    Image Search Results


    a Example of two control and 12 PD LCL lines. Cells were lysed and extracts subjected to quantitative immunoblot analysis with the indicated antibodies and membranes were developed using Odyssey CLx scan Western Blot imaging system. pT73-Rab10 and total Rab10, as well as pS935-LRRK2 and total LRRK2 were multiplexed, and the same control line (S001) was run on every gel to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin, pS935/tubulin, pS935/LRRK2, Rab10/tubulin, pT73-Rab10/tubulin and pT73-Rab10/Rab10 as indicated, with no differences observed between PD LCL lines with or without a centrosome splitting phenotype. Bars represent mean ± s.e.m. c Spearman correlation analysis between levels of LRRK2/tubulin and pT73-Rab10/tubulin (top) or pS935/tubulin and pT73-Rab10/tubulin (bottom). A significant association is observed between LRRK2 or S935-LRRK2 levels and pT73-Rab10 levels in PD LCLs. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p -values are indicated for each correlation analysis.

    Journal: NPJ Parkinson's Disease

    Article Title: A potential patient stratification biomarker for Parkinson´s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

    doi: 10.1038/s41531-023-00624-8

    Figure Lengend Snippet: a Example of two control and 12 PD LCL lines. Cells were lysed and extracts subjected to quantitative immunoblot analysis with the indicated antibodies and membranes were developed using Odyssey CLx scan Western Blot imaging system. pT73-Rab10 and total Rab10, as well as pS935-LRRK2 and total LRRK2 were multiplexed, and the same control line (S001) was run on every gel to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin, pS935/tubulin, pS935/LRRK2, Rab10/tubulin, pT73-Rab10/tubulin and pT73-Rab10/Rab10 as indicated, with no differences observed between PD LCL lines with or without a centrosome splitting phenotype. Bars represent mean ± s.e.m. c Spearman correlation analysis between levels of LRRK2/tubulin and pT73-Rab10/tubulin (top) or pS935/tubulin and pT73-Rab10/tubulin (bottom). A significant association is observed between LRRK2 or S935-LRRK2 levels and pT73-Rab10 levels in PD LCLs. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p -values are indicated for each correlation analysis.

    Article Snippet: The middle piece was incubated with a mouse monoclonal anti-α-tubulin antibody (1:10´000, Sigma, clone DM1A), and the bottom piece was incubated with a rabbit monoclonal anti-pT73-Rab10 antibody (1:1000, Abcam, ab230261) multiplexed with a mouse monoclonal total Rab10 antibody (1:1000, Sigma, SAB5300028).

    Techniques: Western Blot, Imaging

    a Example of three PD LCL lines with or without treatment with LLOMe (1 mM) and MLi2 (50 nM) for 2 h as indicated. Cells were lysed and extracts subjected to multiplexed immunoblotting with the indicated antibodies. b The percentage of LLOMe-triggered increase in pT73-Rab10/Rab10 levels in the absence or presence of MLi2 was calculated for each LCL line. LLOMe triggers similar increases in pT73-Rab10/Rab10 levels in control and PD LCLs with or without a cohesion phenotype. Bars represent mean ± s.e.m.; ctrl versus ctrl + MLi2 ( p = 0.004); PD (split) versus PD (split) + MLi2 ( p = 0.006); PD (non-split) versus PD (non-split) + MLi2 ( p < 0.001): **** p < 0.001; *** p < 0.005; ** p < 0.01. c Paired t -test analysis of LLOMe-triggered increase in pT73-Rab10/Rab10 levels from each cell line in the absence or presence of MLi2. Note that the LLOMe-triggered increase in pT73-Rab10/Rab10 levels is reduced by MLi2 treatment in most cell lines. d Spearman correlation analysis between the percentage of LLOMe-triggered increase in pT73-Rab10/Rab10 levels versus basal pT73-Rab10/Rab10 levels in the absence of LLOMe treatment. There is a negative correlation between basal pT73-Rab10/Rab10 levels and the efficacy of the LLOMe-mediated increase in pT73-Rab10/Rab10 levels. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p -values are indicated (in italics values without the two outliers).

    Journal: NPJ Parkinson's Disease

    Article Title: A potential patient stratification biomarker for Parkinson´s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

    doi: 10.1038/s41531-023-00624-8

    Figure Lengend Snippet: a Example of three PD LCL lines with or without treatment with LLOMe (1 mM) and MLi2 (50 nM) for 2 h as indicated. Cells were lysed and extracts subjected to multiplexed immunoblotting with the indicated antibodies. b The percentage of LLOMe-triggered increase in pT73-Rab10/Rab10 levels in the absence or presence of MLi2 was calculated for each LCL line. LLOMe triggers similar increases in pT73-Rab10/Rab10 levels in control and PD LCLs with or without a cohesion phenotype. Bars represent mean ± s.e.m.; ctrl versus ctrl + MLi2 ( p = 0.004); PD (split) versus PD (split) + MLi2 ( p = 0.006); PD (non-split) versus PD (non-split) + MLi2 ( p < 0.001): **** p < 0.001; *** p < 0.005; ** p < 0.01. c Paired t -test analysis of LLOMe-triggered increase in pT73-Rab10/Rab10 levels from each cell line in the absence or presence of MLi2. Note that the LLOMe-triggered increase in pT73-Rab10/Rab10 levels is reduced by MLi2 treatment in most cell lines. d Spearman correlation analysis between the percentage of LLOMe-triggered increase in pT73-Rab10/Rab10 levels versus basal pT73-Rab10/Rab10 levels in the absence of LLOMe treatment. There is a negative correlation between basal pT73-Rab10/Rab10 levels and the efficacy of the LLOMe-mediated increase in pT73-Rab10/Rab10 levels. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p -values are indicated (in italics values without the two outliers).

    Article Snippet: The middle piece was incubated with a mouse monoclonal anti-α-tubulin antibody (1:10´000, Sigma, clone DM1A), and the bottom piece was incubated with a rabbit monoclonal anti-pT73-Rab10 antibody (1:1000, Abcam, ab230261) multiplexed with a mouse monoclonal total Rab10 antibody (1:1000, Sigma, SAB5300028).

    Techniques: Western Blot

    a Example of control, R1441G-LRRK2 PD and R1441G-LRRK2 NMC LCL lines in the absence or presence of MLi2 (50 nM, 2 h) as indicated. Cell extracts were subjected to multiplexed quantitative immunoblot analysis with the indicated antibodies, and membranes were developed using Odyssey CLx scan Western Blot imaging system. The same control line (002 M) was run on every gel as an internal standard to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin levels (left) and Rab10/tubulin levels (right). Note large variability in the total LRRK2 levels amongst different LCL lines. Scatter plots represent mean ± s.e.m. c Immunoblots were quantified for pT73-Rab10/Rab10 levels, with no significant differences observed between control and PD LCL lines. Scatter plots represent mean ± s.e.m.; ctrl versus ctrl + MLi2 ( p = 0.004); R1441G-LRRK2 PD versus R1441G-LRRK2 PD + MLi2 ( p = 0.0002). **** p < 0.001; *** p < 0.005. d Spearman correlation analysis between levels of pT73-Rab10/Rab10 and LRRK2/tubulin from all LCL lines analyzed. Rho and p -values are indicated. A significant association is observed between the total levels of LRRK2/tubulin and the levels of pT73-Rab10/Rab10.

    Journal: NPJ Parkinson's Disease

    Article Title: A potential patient stratification biomarker for Parkinson´s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

    doi: 10.1038/s41531-023-00624-8

    Figure Lengend Snippet: a Example of control, R1441G-LRRK2 PD and R1441G-LRRK2 NMC LCL lines in the absence or presence of MLi2 (50 nM, 2 h) as indicated. Cell extracts were subjected to multiplexed quantitative immunoblot analysis with the indicated antibodies, and membranes were developed using Odyssey CLx scan Western Blot imaging system. The same control line (002 M) was run on every gel as an internal standard to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin levels (left) and Rab10/tubulin levels (right). Note large variability in the total LRRK2 levels amongst different LCL lines. Scatter plots represent mean ± s.e.m. c Immunoblots were quantified for pT73-Rab10/Rab10 levels, with no significant differences observed between control and PD LCL lines. Scatter plots represent mean ± s.e.m.; ctrl versus ctrl + MLi2 ( p = 0.004); R1441G-LRRK2 PD versus R1441G-LRRK2 PD + MLi2 ( p = 0.0002). **** p < 0.001; *** p < 0.005. d Spearman correlation analysis between levels of pT73-Rab10/Rab10 and LRRK2/tubulin from all LCL lines analyzed. Rho and p -values are indicated. A significant association is observed between the total levels of LRRK2/tubulin and the levels of pT73-Rab10/Rab10.

    Article Snippet: The middle piece was incubated with a mouse monoclonal anti-α-tubulin antibody (1:10´000, Sigma, clone DM1A), and the bottom piece was incubated with a rabbit monoclonal anti-pT73-Rab10 antibody (1:1000, Abcam, ab230261) multiplexed with a mouse monoclonal total Rab10 antibody (1:1000, Sigma, SAB5300028).

    Techniques: Western Blot, Imaging

    Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of Rab10 phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).

    Journal: ACS Chemical Neuroscience

    Article Title: Doubly Constrained C-terminal of Roc (COR) Domain-Derived Peptides Inhibit Leucine-Rich Repeat Kinase 2 (LRRK2) Dimerization

    doi: 10.1021/acschemneuro.3c00259

    Figure Lengend Snippet: Doubly constrained peptides downregulate LRRK2 dimerization and LRRK2 kinase activity and do not cause microtubule mislocalization of LRRK2. (a) Mass photometry was applied to determine the molecular weight of LRRK2 to measure monomer and dimer species. Purified full-length LRRK2 was incubated with 7.5-fold excess of peptide for 30 min at 30 °C. The percentage of dimer for the different conditions is depicted. Full diagrams are provided in Figure S9 . * p < 0.05, ** p < 0.01. (b) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate; ns not significant, * p < 0.05, ** p < 0.005, **** p ≤ 0.0001. (c) Western blot analysis of Rab10 phosphorylation in A549 PPM1H knockout cells treated with different concentrations of doubly constrained peptides demonstrates downregulation of LRRK2 kinase activity as compared to the untreated control after incubation for 6 h. (d) Quantification of mean intensity of phospho-Rab10 signal normalized to GAPDH over total Rab10 signal normalized to GAPDH, with standard error of mean (SEM) for at least three independent experiments. An unpaired 2-tailed t test was performed; **** p ≤ 0.0001, ns not significant ( p > 0.05). (e) HEK293 cells were transfected with GFP–LRRK2 for 24 h and subsequently treated with DMSO, 1 μM MLi-2, or 2.5 μM ECOR or ECOR A12G peptide for 6 h at 37 °C. The cells were treated with media containing SiR-tubulin for tubulin staining and verapamil (a broad-spectrum efflux pump inhibitor). The cells treated with MLi-2 show skein-like filamentous structures that overlap with tubulin filaments stained by SiR-tubulin. Scale bar 10 μm. (f) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in panel c. The graph represents the average percentage of cells, and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p ≤ 0.0001, ns not significant ( p > 0.05).

    Article Snippet: The membranes were blocked in nonfat milk and probed with Rab10 (phospho-T73) (Abcam; ab241060, Lot; GR327 4620-4), total Rab10 (Cell Signaling Technology; #4262S), and total GAPDH (14C10) (Cell Signaling Technology; #2118S) antibodies overnight at 4 °C, followed by incubation with HRP anti-rabbit secondary antibody, and developed using ECL.

    Techniques: Activity Assay, Molecular Weight, Purification, Incubation, Cell Surface Biotinylation Assay, Western Blot, Knock-Out, Transfection, Staining

    Neutrophils were isolated from 12 healthy donors and treated with or without 100 nM MLi-2 for 30 min. ( A ) Viability (DAPI) and purity (CD66b) of neutrophils isolated from whole blood of 12 healthy volunteers were assessed via flow cytometer analysis. ( B and C ) Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. Similar results were obtained in two independent experiments. Quantitation of immunoblots was undertaken by analysing LRRK2 (full length)/GAPDH ratio ( Bi ), total LRRK2 (170 kDa species)/GAPDH ratio ( Bii ), phospho-Ser935 LRRK2 (full length)/total LRRK2 (full length) ratio ( Biii ) and phospho-Ser935 LRRK2 (170 kDa species)/total LRRK2 (170 kDa species) ratio ( Biv ), total Rab10/GAPDH ratio ( Ci ), phospho-Thr73 Rab10/GAPDH ratio ( Cii ) and phospho-Thr73 Rab10/total Rab10 ratio ( Ciii ).

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: Neutrophils were isolated from 12 healthy donors and treated with or without 100 nM MLi-2 for 30 min. ( A ) Viability (DAPI) and purity (CD66b) of neutrophils isolated from whole blood of 12 healthy volunteers were assessed via flow cytometer analysis. ( B and C ) Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. Similar results were obtained in two independent experiments. Quantitation of immunoblots was undertaken by analysing LRRK2 (full length)/GAPDH ratio ( Bi ), total LRRK2 (170 kDa species)/GAPDH ratio ( Bii ), phospho-Ser935 LRRK2 (full length)/total LRRK2 (full length) ratio ( Biii ) and phospho-Ser935 LRRK2 (170 kDa species)/total LRRK2 (170 kDa species) ratio ( Biv ), total Rab10/GAPDH ratio ( Ci ), phospho-Thr73 Rab10/GAPDH ratio ( Cii ) and phospho-Thr73 Rab10/total Rab10 ratio ( Ciii ).

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Isolation, Flow Cytometry, Western Blot, Imaging, Quantitation Assay

    ( A ) Both neutrophils and PBMCs were prepared from the same six healthy donors and treated with or without 100 nM MLi-2 for 30 min. Cells were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. ( B ) Quantitation of immunoblots was undertaken analysing total LRRK2 (full length)/GAPDH ( Bi ), total Rab10/GAPDH ratio ( Bii ), phospho-Ser935 LRRK2 (full length)/total LRRK2 (full length) ( Biii ) and total phospho-Thr73 Rab10/total Rab10 ( Biv ).

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: ( A ) Both neutrophils and PBMCs were prepared from the same six healthy donors and treated with or without 100 nM MLi-2 for 30 min. Cells were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. ( B ) Quantitation of immunoblots was undertaken analysing total LRRK2 (full length)/GAPDH ( Bi ), total Rab10/GAPDH ratio ( Bii ), phospho-Ser935 LRRK2 (full length)/total LRRK2 (full length) ( Biii ) and total phospho-Thr73 Rab10/total Rab10 ( Biv ).

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Western Blot, Imaging, Quantitation Assay

    Neutrophils were isolated from 12 healthy donors and treated with or without 100 nM MLi-2 for 30 min. Cells were then lysed and 10 µg of whole cell extract subjected to electrophoresis in which the Phos-tag acrylamide was polymerised into the polyacrylamide gel in order to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. The gels were subjected to immunoblot analysis with an antibody that specifically recognises Rab10 (1 µg/ml antibody), and the band corresponding to phosphorylated and non-phosphorylated Rab10 is marked with open and filled circles, respectively. A low (top panel), medium (middle panel) and high exposure (bottom panel) of the immunoblot are shown. Similar results were obtained in two separate experiments.

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: Neutrophils were isolated from 12 healthy donors and treated with or without 100 nM MLi-2 for 30 min. Cells were then lysed and 10 µg of whole cell extract subjected to electrophoresis in which the Phos-tag acrylamide was polymerised into the polyacrylamide gel in order to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. The gels were subjected to immunoblot analysis with an antibody that specifically recognises Rab10 (1 µg/ml antibody), and the band corresponding to phosphorylated and non-phosphorylated Rab10 is marked with open and filled circles, respectively. A low (top panel), medium (middle panel) and high exposure (bottom panel) of the immunoblot are shown. Similar results were obtained in two separate experiments.

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Isolation, Electrophoresis, Western Blot

    The study utilised pure populations of immune cells isolated from human blood using fluorescence-activated cell sorting, and whole cell proteomics data were generated. Data were analysed using the histone ruler to estimate protein copy numbers per cell. The graphs show the number of protein copies per cell for LRRK2 and RAB10 in a range of peripheral blood immune cells including subsets of T cells, B cells, monocytes, NK cells, dendritic cells and the granulocytes neutrophils, basophils and eosinophils.

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: The study utilised pure populations of immune cells isolated from human blood using fluorescence-activated cell sorting, and whole cell proteomics data were generated. Data were analysed using the histone ruler to estimate protein copy numbers per cell. The graphs show the number of protein copies per cell for LRRK2 and RAB10 in a range of peripheral blood immune cells including subsets of T cells, B cells, monocytes, NK cells, dendritic cells and the granulocytes neutrophils, basophils and eosinophils.

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Isolation, Fluorescence, FACS, Generated

    The immunoblots from were quantified for phospho-Thr73 Rab10/GAPDH ratio ( A ), phospho-Thr73 Rab10/total Rab10 ratio ( B ), LRRK2-dependent phospho-Thr73 Rab10/GAPDH ratio ( C ) and LRRK2-dependent phospho-Thr73 Rab10/total Rab10 ratio ( D ). Data were analysed by one-way ANOVA with Bonferroni's multiple comparisons test. Data presented as means ± SD; * P < 0.05, ** P < 0.005.

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: The immunoblots from were quantified for phospho-Thr73 Rab10/GAPDH ratio ( A ), phospho-Thr73 Rab10/total Rab10 ratio ( B ), LRRK2-dependent phospho-Thr73 Rab10/GAPDH ratio ( C ) and LRRK2-dependent phospho-Thr73 Rab10/total Rab10 ratio ( D ). Data were analysed by one-way ANOVA with Bonferroni's multiple comparisons test. Data presented as means ± SD; * P < 0.05, ** P < 0.005.

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Western Blot

    The immunoblots from were quantified for full-length LRRK2/GAPDH ratio ( A ), ∼170 kDa species of LRRK2/GAPDH ratio ( B ), phospho-Ser935 LRRK2 (full length)/total LRRK2 (full length) ratio ( C ) and total Rab10/GAPDH ratio ( D ).

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: The immunoblots from were quantified for full-length LRRK2/GAPDH ratio ( A ), ∼170 kDa species of LRRK2/GAPDH ratio ( B ), phospho-Ser935 LRRK2 (full length)/total LRRK2 (full length) ratio ( C ) and total Rab10/GAPDH ratio ( D ).

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Western Blot

    ( A ) Neutrophils were isolated from two healthy donors and treated with the indicated concentrations of MLi-2 or PF-06447475 for 30 min. Cells were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. ( B ) Quantitation of immunoblots was undertaken by analysing phospho-Thr73 Rab10/total Rab10 ratio ( Bi ) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio ( Bii ). ( C and D ) Quantification of immunoblots as above, phospho-Thr73 Rab10/total Rab10 ratio ( Di ) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio ( Dii ), except that neutrophils were isolated from two healthy donors and treated with MLi-2 at concentrations of 100 nM for the indicated times. Similar results were obtained in two independent experiments.

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: ( A ) Neutrophils were isolated from two healthy donors and treated with the indicated concentrations of MLi-2 or PF-06447475 for 30 min. Cells were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. ( B ) Quantitation of immunoblots was undertaken by analysing phospho-Thr73 Rab10/total Rab10 ratio ( Bi ) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio ( Bii ). ( C and D ) Quantification of immunoblots as above, phospho-Thr73 Rab10/total Rab10 ratio ( Di ) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio ( Dii ), except that neutrophils were isolated from two healthy donors and treated with MLi-2 at concentrations of 100 nM for the indicated times. Similar results were obtained in two independent experiments.

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Isolation, Western Blot, Imaging, Quantitation Assay

    Blood was drawn from three healthy donors and left unprocessed at room temperature. At the indicated times, neutrophils were isolated from the blood and treated with or without MLi-2 at 100 nM for 30 min. ( A ) Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. Similar results were obtained in two independent experiments. ( B ) Quantitation of immunoblots was undertaken by analysing phospho-Thr73 Rab10/total Rab10 ratio (top panel) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio (bottom panel).

    Journal: Biochemical Journal

    Article Title: Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

    doi: 10.1042/BCJ20170803

    Figure Lengend Snippet: Blood was drawn from three healthy donors and left unprocessed at room temperature. At the indicated times, neutrophils were isolated from the blood and treated with or without MLi-2 at 100 nM for 30 min. ( A ) Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. Similar results were obtained in two independent experiments. ( B ) Quantitation of immunoblots was undertaken by analysing phospho-Thr73 Rab10/total Rab10 ratio (top panel) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio (bottom panel).

    Article Snippet: The MJFF-total Rab10 mouse monoclonal antibody was similarly sensitive and clean as the rabbit anti-Rab10 antibody from Cell Signaling Technology (#8127) used at a 1 : 1000 dilution.

    Techniques: Isolation, Western Blot, Imaging, Quantitation Assay