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Millipore rabbit polyclonal anti-rab8a

RAB7L1 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous <t>RAB8A</t> in the presence of wildtype LRRK2. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 (wt) (green), mRFP-tagged RAB7L1 (red), or a combination thereof, treated with 100 nM MLi2 for 60 min where indicated, and stained with RAB8A (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and with pericentrin antibody (Alexa 405-conjugated secondary antibody, pseudo-colored in gray). Scale bar, 10 μm. (B) Quantification of the percentage of transfected cells displaying pericentrosomal/centrosomal RAB8A staining of the type of experiments depicted in (A) . At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Rabbit Polyclonal Anti Rab8a, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A"

Article Title: RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00417

RAB7L1 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous RAB8A in the presence of wildtype LRRK2. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 (wt) (green), mRFP-tagged RAB7L1 (red), or a combination thereof, treated with 100 nM MLi2 for 60 min where indicated, and stained with RAB8A (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and with pericentrin antibody (Alexa 405-conjugated secondary antibody, pseudo-colored in gray). Scale bar, 10 μm. (B) Quantification of the percentage of transfected cells displaying pericentrosomal/centrosomal RAB8A staining of the type of experiments depicted in (A) . At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Figure Legend Snippet: RAB7L1 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous RAB8A in the presence of wildtype LRRK2. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 (wt) (green), mRFP-tagged RAB7L1 (red), or a combination thereof, treated with 100 nM MLi2 for 60 min where indicated, and stained with RAB8A (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and with pericentrin antibody (Alexa 405-conjugated secondary antibody, pseudo-colored in gray). Scale bar, 10 μm. (B) Quantification of the percentage of transfected cells displaying pericentrosomal/centrosomal RAB8A staining of the type of experiments depicted in (A) . At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Techniques Used: Transfection, Staining

Coexpression of RAB7L1 and wildtype LRRK2 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous phospho-RAB8A. (A) Example of HEK293T cells transfected with GFP-tagged wildtype LRRK2 (wt) (green), mRFP-tagged RAB7L1 (red), or a combination thereof, and stained with pericentrin (Alexa 405-conjugated secondary antibody, pseudo-colored in gray), and an anti-phospho-T72-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), the antibody preabsorbed with phospho-peptide (p-peptide), or cells treated with 100 nM MLi2 for 60 min prior to immunocytochemistry, as indicated. Scale bar, 10 μm. (B) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining from experiments of the type depicted in (A) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (C) HEK293T cells were transfected with the different constructs and treated with 100 nM MLi2 for 60 min where indicated, and extracts blotted for phospho-RAB8A, total RAB8A, GFP (for LRRK2 detection), flag (for RAB7L1 detection), and GAPDH or tubulin as loading controls.

Figure Legend Snippet: Coexpression of RAB7L1 and wildtype LRRK2 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous phospho-RAB8A. (A) Example of HEK293T cells transfected with GFP-tagged wildtype LRRK2 (wt) (green), mRFP-tagged RAB7L1 (red), or a combination thereof, and stained with pericentrin (Alexa 405-conjugated secondary antibody, pseudo-colored in gray), and an anti-phospho-T72-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), the antibody preabsorbed with phospho-peptide (p-peptide), or cells treated with 100 nM MLi2 for 60 min prior to immunocytochemistry, as indicated. Scale bar, 10 μm. (B) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining from experiments of the type depicted in (A) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (C) HEK293T cells were transfected with the different constructs and treated with 100 nM MLi2 for 60 min where indicated, and extracts blotted for phospho-RAB8A, total RAB8A, GFP (for LRRK2 detection), flag (for RAB7L1 detection), and GAPDH or tubulin as loading controls.

Techniques Used: Transfection, Staining, Immunocytochemistry, Construct

Coexpression of RAB7L1 with ankyrin domain residue mutant LRRK2 does not cause centrosomal deficits or pericentrosomal/centrosomal accumulation of endogenous phospho-RAB8A. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 or LD-LRRK2 (green) in the presence or absence of mRFP-tagged RAB7L1 (red) as indicated, and stained with pericentrin antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (B) Quantification of the split centrosome phenotype in non-transfected cells or cells expressing wildtype LRRK2 or LD-LRRK2 in the presence or absence of RAB7L1 as indicated. At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (C) Quantification of recruitment of either wildtype LRRK2 or LD-LRRK2 to mRFP-RAB7L1 compartment. Around 100 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (D) Example of HEK293T cells transfected with constructs as indicated, and stained with RAB8A antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (E) Quantification of the percentage of non-transfected or transfected cells displaying pericentrosomal/centrosomal RAB8A staining of the type of experiments depicted in (D) . At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM. ( n = 3 experiments); ∗∗∗∗ p < 0.001. (F) Example of HEK293T cells transfected with constructs as indicated, and stained with an anti-phospho-T72-RAB8A antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (G) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining from experiments of the type depicted in (F) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (H) HEK293T cells were transfected with the indicated constructs, and extracts blotted for phospho-RAB8A, GFP (for LRRK2 detection), flag (for RAB7L1 detection), and tubulin as loading control.

Figure Legend Snippet: Coexpression of RAB7L1 with ankyrin domain residue mutant LRRK2 does not cause centrosomal deficits or pericentrosomal/centrosomal accumulation of endogenous phospho-RAB8A. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 or LD-LRRK2 (green) in the presence or absence of mRFP-tagged RAB7L1 (red) as indicated, and stained with pericentrin antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (B) Quantification of the split centrosome phenotype in non-transfected cells or cells expressing wildtype LRRK2 or LD-LRRK2 in the presence or absence of RAB7L1 as indicated. At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (C) Quantification of recruitment of either wildtype LRRK2 or LD-LRRK2 to mRFP-RAB7L1 compartment. Around 100 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (D) Example of HEK293T cells transfected with constructs as indicated, and stained with RAB8A antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (E) Quantification of the percentage of non-transfected or transfected cells displaying pericentrosomal/centrosomal RAB8A staining of the type of experiments depicted in (D) . At least 50 transfected cells were analyzed per condition per experiment. Bars represent mean ± SEM. ( n = 3 experiments); ∗∗∗∗ p < 0.001. (F) Example of HEK293T cells transfected with constructs as indicated, and stained with an anti-phospho-T72-RAB8A antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (G) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining from experiments of the type depicted in (F) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (H) HEK293T cells were transfected with the indicated constructs, and extracts blotted for phospho-RAB8A, GFP (for LRRK2 detection), flag (for RAB7L1 detection), and tubulin as loading control.

Techniques Used: Mutagenesis, Transfection, Staining, Fluorescence, Expressing, Construct

Integrity of the Golgi complex is required for the RAB7L1-mediated phospho-RAB8A accumulation and centrosomal cohesion deficits in the presence of wildtype LRRK2. (A) HEK293T cells were cotransfected with GFP-tagged LRRK2 (green) and mRFP-tagged RAB7L1 (red), and either treated or non-treated with brefeldin A (BFA; 7.5 μg/ml) for 3 h as indicated before staining with a knockout-validated rabbit monoclonal total RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and DAPI (pseudo-colored in gray). Scale bar, 10 μm. (B) Quantification of the percentage of non-transfected or transfected cells displaying pericentrosomal/centrosomal RAB8A staining, in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (A) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001; ∗∗∗ p < 0.005. (C) HEK293T cells were transfected as indicated, and either treated or non-treated with brefeldin A as described above, before staining with a rabbit polyclonal phospho-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and DAPI (pseudo-colored in gray). Scale bar, 10 μm. (D) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining, in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (C) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (E) Cells were transfected as indicated, and either treated or non-treated with brefeldin A as described above, before staining with pericentrin antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and DAPI (pseudo-colored in gray). Scale bar, 10 μm. (F) Quantification of the split centrosome phenotype in non-transfected or transfected cells as indicated, in the presence or absence of brefeldin A, from experiments of the type depicted in (E) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Figure Legend Snippet: Integrity of the Golgi complex is required for the RAB7L1-mediated phospho-RAB8A accumulation and centrosomal cohesion deficits in the presence of wildtype LRRK2. (A) HEK293T cells were cotransfected with GFP-tagged LRRK2 (green) and mRFP-tagged RAB7L1 (red), and either treated or non-treated with brefeldin A (BFA; 7.5 μg/ml) for 3 h as indicated before staining with a knockout-validated rabbit monoclonal total RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and DAPI (pseudo-colored in gray). Scale bar, 10 μm. (B) Quantification of the percentage of non-transfected or transfected cells displaying pericentrosomal/centrosomal RAB8A staining, in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (A) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001; ∗∗∗ p < 0.005. (C) HEK293T cells were transfected as indicated, and either treated or non-treated with brefeldin A as described above, before staining with a rabbit polyclonal phospho-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and DAPI (pseudo-colored in gray). Scale bar, 10 μm. (D) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining, in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (C) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (E) Cells were transfected as indicated, and either treated or non-treated with brefeldin A as described above, before staining with pericentrin antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue) and DAPI (pseudo-colored in gray). Scale bar, 10 μm. (F) Quantification of the split centrosome phenotype in non-transfected or transfected cells as indicated, in the presence or absence of brefeldin A, from experiments of the type depicted in (E) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Techniques Used: Staining, Knock-Out, Transfection

Integrity of the Golgi complex is not required for the pathogenic LRRK2-mediated phospho-RAB8A accumulation and centrosomal cohesion deficits. (A) HEK293T cells were transfected with Y1699C-mutant LRRK2 (green), either treated or non-treated with brefeldin A (BFA; 7.5 μg/ml) for 3 h, and stained with a knockout-validated rabbit monoclonal anti-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), a trans -Golgi marker antibody (Golgin245, Alexa 594-conjugated secondary antibody, red) and DAPI (pseudo-colored in cyan). Scale bar, 10 μm. (B) Quantification of the percentage of non-transfected cells, or cells transfected with the various pathogenic LRRK2 constructs displaying pericentrosomal/centrosomal RAB8A staining in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (A) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001; ∗∗∗ p < 0.005. (C) Cells were transfected with Y1699C-mutant LRRK2 (green), and either treated or non-treated with brefeldin A as described above before staining with a rabbit polyclonal phospho-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), a trans -Golgi marker antibody (Golgin245, Alexa 594-conjugated secondary antibody, red) and DAPI (pseudo-colored in cyan). Scale bar, 10 μm. (D) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining, in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (C) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (E) Cells were transfected with Y1699C-mutant LRRK2 (green), and either treated or non-treated with brefeldin A as described above before staining with a pericentrin antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), a trans -Golgi marker antibody (Golgin245, Alexa 594-conjugated secondary antibody, red) and DAPI (pseudo-colored in cyan). Scale bar, 10 μm. (F) Quantification of the split centrosome phenotype in non-transfected or transfected cells as indicated, in the presence or absence of brefeldin A, from experiments of the type depicted in (E) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Figure Legend Snippet: Integrity of the Golgi complex is not required for the pathogenic LRRK2-mediated phospho-RAB8A accumulation and centrosomal cohesion deficits. (A) HEK293T cells were transfected with Y1699C-mutant LRRK2 (green), either treated or non-treated with brefeldin A (BFA; 7.5 μg/ml) for 3 h, and stained with a knockout-validated rabbit monoclonal anti-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), a trans -Golgi marker antibody (Golgin245, Alexa 594-conjugated secondary antibody, red) and DAPI (pseudo-colored in cyan). Scale bar, 10 μm. (B) Quantification of the percentage of non-transfected cells, or cells transfected with the various pathogenic LRRK2 constructs displaying pericentrosomal/centrosomal RAB8A staining in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (A) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001; ∗∗∗ p < 0.005. (C) Cells were transfected with Y1699C-mutant LRRK2 (green), and either treated or non-treated with brefeldin A as described above before staining with a rabbit polyclonal phospho-RAB8A antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), a trans -Golgi marker antibody (Golgin245, Alexa 594-conjugated secondary antibody, red) and DAPI (pseudo-colored in cyan). Scale bar, 10 μm. (D) Quantification of the percentage of non-transfected or transfected cells displaying phospho-RAB8A staining, in the presence or absence of brefeldin A as indicated, from experiments of the type depicted in (C) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001. (E) Cells were transfected with Y1699C-mutant LRRK2 (green), and either treated or non-treated with brefeldin A as described above before staining with a pericentrin antibody (Alexa 647-conjugated secondary antibody, pseudo-colored in blue), a trans -Golgi marker antibody (Golgin245, Alexa 594-conjugated secondary antibody, red) and DAPI (pseudo-colored in cyan). Scale bar, 10 μm. (F) Quantification of the split centrosome phenotype in non-transfected or transfected cells as indicated, in the presence or absence of brefeldin A, from experiments of the type depicted in (E) . At least 50 cells were analyzed per condition per experiment. Bars represent mean ± SEM ( n = 3 experiments); ∗∗∗∗ p < 0.001.

Techniques Used: Transfection, Mutagenesis, Staining, Knock-Out, Marker, Construct

Knockdown of RAB8A significantly reverses the centrosomal cohesion deficits mediated by RAB7L1 and wildtype LRRK2. (A) Representative Western blot of extracts from control pCMV-transfected cells, or cells transfected with wildtype (wt) LRRK2 and RAB7L1, along with either ctrl-siRNA or RAB8A-siRNA as indicated, and blotted against RAB8A or tubulin as loading control. (B) Quantification of the type of experiments depicted in (A) , with levels of RAB8A normalized to tubulin and to RAB8A levels in the presence of ctrl-siRNA. Bars represent ± SEM ( n = 3 experiments); ∗ p < 0.05. (C) Example of HEK293T cells co-transfected with GFP-tagged wildtype LRRK2 (green) and mRFP-tagged RAB7L1 (red), and either with ctrl-siRNA or RAB8A-siRNA as indicated, and stained with pericentrin antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (D) Quantification of the split centrosome phenotype in control cells transfected with either ctrl-siRNA or RAB8A-siRNA, or in cells co-transfected with the respective siRNAs and with wildtype LRRK2 and RAB7L1. Around 100 transfected cells were analyzed per condition per experiment. Bars represent ± SEM ( n = 3 experiments); ∗∗∗ p < 0.005; ∗∗ p < 0.01.

Figure Legend Snippet: Knockdown of RAB8A significantly reverses the centrosomal cohesion deficits mediated by RAB7L1 and wildtype LRRK2. (A) Representative Western blot of extracts from control pCMV-transfected cells, or cells transfected with wildtype (wt) LRRK2 and RAB7L1, along with either ctrl-siRNA or RAB8A-siRNA as indicated, and blotted against RAB8A or tubulin as loading control. (B) Quantification of the type of experiments depicted in (A) , with levels of RAB8A normalized to tubulin and to RAB8A levels in the presence of ctrl-siRNA. Bars represent ± SEM ( n = 3 experiments); ∗ p < 0.05. (C) Example of HEK293T cells co-transfected with GFP-tagged wildtype LRRK2 (green) and mRFP-tagged RAB7L1 (red), and either with ctrl-siRNA or RAB8A-siRNA as indicated, and stained with pericentrin antibody (Alexa 405-conjugated secondary antibody, blue) and TO-PRO-3 (far red fluorescence similar to Alexa 647, pseudo-colored in cyan). Scale bar, 10 μm. (D) Quantification of the split centrosome phenotype in control cells transfected with either ctrl-siRNA or RAB8A-siRNA, or in cells co-transfected with the respective siRNAs and with wildtype LRRK2 and RAB7L1. Around 100 transfected cells were analyzed per condition per experiment. Bars represent ± SEM ( n = 3 experiments); ∗∗∗ p < 0.005; ∗∗ p < 0.01.

Techniques Used: Western Blot, Transfection, Staining, Fluorescence

Model for how altered RAB7L1 levels and pathogenic LRRK2 cause centrosomal alterations via RAB8A phosphorylation. Under normal conditions, wildtype LRRK2 is largely cytosolic. Under conditions of increased RAB7L1 levels (increased RAB7L1), wildtype LRRK2 becomes recruited to the Golgi apparatus, where it phosphorylates Golgi-resident RAB8A. Phosphorylated RAB8A accumulates at/around the centrosome, possibly via binding to RILPL1/RILPL2, resulting in centrosomal cohesion deficits. Pathogenic LRRK2 (mutant LRRK2) displays a more pericentrosomal/centrosomal localization by currently unknown mechanisms, but possibly involving enhanced binding to RAB8A on the early recycling compartment and/or perinuclear vesicular structures. This causes RAB8A phosphorylation in those compartments, with phosphorylated RAB8A then accumulating at/around the centrosome to cause centrosomal cohesion deficits in a manner independent on Rab7L1 or on Golgi integrity. ER, endoplasmic reticulum; ERC, early recycling compartment.

Figure Legend Snippet: Model for how altered RAB7L1 levels and pathogenic LRRK2 cause centrosomal alterations via RAB8A phosphorylation. Under normal conditions, wildtype LRRK2 is largely cytosolic. Under conditions of increased RAB7L1 levels (increased RAB7L1), wildtype LRRK2 becomes recruited to the Golgi apparatus, where it phosphorylates Golgi-resident RAB8A. Phosphorylated RAB8A accumulates at/around the centrosome, possibly via binding to RILPL1/RILPL2, resulting in centrosomal cohesion deficits. Pathogenic LRRK2 (mutant LRRK2) displays a more pericentrosomal/centrosomal localization by currently unknown mechanisms, but possibly involving enhanced binding to RAB8A on the early recycling compartment and/or perinuclear vesicular structures. This causes RAB8A phosphorylation in those compartments, with phosphorylated RAB8A then accumulating at/around the centrosome to cause centrosomal cohesion deficits in a manner independent on Rab7L1 or on Golgi integrity. ER, endoplasmic reticulum; ERC, early recycling compartment.

Techniques Used: Binding Assay, Mutagenesis

Knock-Out:

Article Title: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation
Article Snippet: Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal anti-γ-tubulin (Abcam, ab11316, 1:1000), mouse monoclonal anti-c-Myc (Sigma, clone 9E10, M4439 1:500), mouse monoclonal anti-flag (Sigma, clone M2, 1:500), rabbit polyclonal anti-Rab8a (Millipore, ABC423, 1:1000), mouse anti-Golgin97 (Molecular Probes, A-21270, 1:100), rabbit polyclonal anti-β-COP (Invitrogen, PA1–061, 1:750), and mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611,280, 1:400).

Article Title: RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A
Article Snippet: Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611280, 1:400), rabbit polyclonal anti-RAB8A (Millipore, ABC423, 1:1000), knockout-validated rabbit monoclonal anti-RAB8A (Abcam, ab188574, 1:1000), rabbit polyclonal anti-T72-phospho-RAB8A (1:500, generous gift of D. Alessi, University of Dundee, United Kingdom), knockout-validated sheep polyclonal anti-RAB8A (S969D) and sheep polyclonal anti-T72-phospho-RAB8A (S874D) (MRC PPU).

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