mouse monoclonal anti rab8a  (Novus Biologicals)

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Active RAB8A and Rabin8 rescue the LRRK2-mediated deficit in EGF binding and degradation. A , HeLa cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB8A constructs followed by quantification of the amount of surface-bound fluorescent EGF. n = 4 independent experiments. *, p < 0.05. B , cells were transfected as indicated followed by quantification of internalized Alexa555-EGF in transfected cells after 10 ( left ) and 30 min ( right ) of internalization. Values are normalized to the amount of Alexa555-EGF binding at t = 0. n = 4 independent experiments. *, p < 0.05. C , cells were cotransfected with G2019S LRRK2 and the indicated RAB8A constructs, and surface-bound fluorescent EGF was quantified. n = 8 independent experiments. *, p < 0.05. D , cells were cotransfected with G2019S LRRK2 and the indicated RAB8A constructs followed by quantification of internalized Alexa555-EGF after 10 ( left ) and 30 min ( right ) of internalization. n = 8 independent experiments. ****, p < 0.001. E , cells were transfected with either empty pCMV vector ( ctrl ) or with Rabin8, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. F , cells were transfected as indicated followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. G , cells were transfected with either empty pCMV vector ( ctrl ) or cotransfected with G2019S pathogenic LRRK2 and either pCMV vector or Rabin8 as indicated, and surface-bound fluorescent EGF was quantified. n = 3 experiments. *, p < 0.05. H , cells were transfected as indicated followed by quantification of internalized fluorescent EGF as described above. n = 3 independent experiments. ***, p < 0.005. All error bars represent S.E.M.

Article Snippet: Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP (1:2000; Abcam, ab6556), a mouse monoclonal anti-GAPDH (1:2000; Abcam, ab9484), a mouse monoclonal anti-LRRK2 (1:1000; NeuroMab, 75-253), a mouse monoclonal anti-RAB8A antibody (1:1000; BD Biosciences, 610844), a mouse monoclonal anti-tubulin antibody (clone DM1A; 1:10,000; Sigma), a sheep polyclonal anti-RAB8A antibody (1:500; S969D, MRC-PPU Reagents), a rabbit polyclonal anti-RAB7 antibody (1:1000; Sigma, R4779), a mouse monoclonal anti-RAB11 antibody (1:1000; BD Biosciences, 610656), and a knockout-validated rabbit monoclonal anti-RAB4 antibody (1:1000; Abcam, ab109009).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Construct

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Phosphodeficient RAB8A, but not WT or phosphomimetic RAB8A variants, revert the LRRK2-mediated effects on EGFR trafficking. A , HeLa cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB8A constructs, and surface-bound fluorescent EGF was quantified. n = 4 independent experiments. *, p < 0.05. B , cells were transfected with the indicated constructs followed by quantification of internalized fluorescent EGF. n = 4 independent experiments. *, p < 0.05. C , cells were cotransfected with G2019S LRRK2 and the indicated RAB8A constructs, and surface-bound fluorescent EGF was quantified. n = 8 independent experiments. *, p < 0.05. D , cells were transfected with the indicated constructs, and internalized fluorescent EGF was quantified at 10 ( left ) and 30 min ( right ). n = 8 independent experiments. *, p < 0.05; ***, p < 0.005; ****, p < 0.001. All error bars represent S.E.M.

Article Snippet: Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP (1:2000; Abcam, ab6556), a mouse monoclonal anti-GAPDH (1:2000; Abcam, ab9484), a mouse monoclonal anti-LRRK2 (1:1000; NeuroMab, 75-253), a mouse monoclonal anti-RAB8A antibody (1:1000; BD Biosciences, 610844), a mouse monoclonal anti-tubulin antibody (clone DM1A; 1:10,000; Sigma), a sheep polyclonal anti-RAB8A antibody (1:500; S969D, MRC-PPU Reagents), a rabbit polyclonal anti-RAB7 antibody (1:1000; Sigma, R4779), a mouse monoclonal anti-RAB11 antibody (1:1000; BD Biosciences, 610656), and a knockout-validated rabbit monoclonal anti-RAB4 antibody (1:1000; Abcam, ab109009).

Techniques: Transfection, Plasmid Preparation, Construct

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Knockdown of RAB8A mimics the endolysosomal trafficking deficits mediated by G2019S LRRK2. A , HeLa cells were either nontransfected (−) or transfected with ctrl-siRNA or RAB8A-siRNA, and cell extracts (30 μg) were analyzed by Western blotting for RAB8A protein levels and tubulin as a loading control. B , quantification of the type of experiments depicted in A . RAB8A levels in the presence of RAB8A-siRNA were normalized to levels in the presence of ctrl-siRNA. n = 3 independent experiments. *, p < 0.05. C , cells were either left untreated (−) or transfected with ctrl-siRNA or RAB8A-siRNA, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. D , cells were either left untreated (−) or transfected with ctrl-siRNA or RAB8A-siRNA followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. **, p < 0.01; ****, p < 0.001. E , cells were either left untreated or cotransfected with ctrl-siRNA or RAB8A-siRNA in the absence or presence of GFP-tagged active RAB7A (RAB7A-Q67L), and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. F , cells were either left untreated or cotransfected with ctrl-siRNA or RAB8A-siRNA in the absence or presence of RAB7A-Q67L, and internalized fluorescent EGF was quantified at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. G , cells were either treated with ctrl-siRNA or RAB8A-siRNA as indicated, and the RAB7-binding domain of RILP coupled to GST was used to pull down the GTP-bound form of RAB7 from cell lysates (300 μg). Input (10%) was run alongside pulldowns to demonstrate equal levels of total RAB7 protein in ctrl-siRNA– or RAB8A-siRNA–treated cells, and the levels of RAB8A and tubulin were analyzed on a separate gel. H , experiments of the type depicted in G were quantified, and the amount of RAB7 isolated by GST-RILP was expressed relative to input. n = 3 independent experiments. ***, p < 0.005. I , cells were either treated with ctrl-siRNA or RAB8A-siRNA as indicated, and a conformation-specific antibody was used to immunoprecipitate active RAB7 from cell lysates (2 mg). As a positive control, ctrl-siRNA–treated cell extracts were incubated with 100 μ m GTPγS to activate RAB7A before immunoprecipitation. Input (1%) was run alongside pulldowns to demonstrate equal levels of total RAB7 protein in ctrl-siRNA– or RAB8A-siRNA–treated cells, and the levels of RAB8A and tubulin were analyzed on a separate gel. All error bars represent S.E.M.

Article Snippet: Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP (1:2000; Abcam, ab6556), a mouse monoclonal anti-GAPDH (1:2000; Abcam, ab9484), a mouse monoclonal anti-LRRK2 (1:1000; NeuroMab, 75-253), a mouse monoclonal anti-RAB8A antibody (1:1000; BD Biosciences, 610844), a mouse monoclonal anti-tubulin antibody (clone DM1A; 1:10,000; Sigma), a sheep polyclonal anti-RAB8A antibody (1:500; S969D, MRC-PPU Reagents), a rabbit polyclonal anti-RAB7 antibody (1:1000; Sigma, R4779), a mouse monoclonal anti-RAB11 antibody (1:1000; BD Biosciences, 610656), and a knockout-validated rabbit monoclonal anti-RAB4 antibody (1:1000; Abcam, ab109009).

Techniques: Transfection, Western Blot, Binding Assay, Isolation, Positive Control, Incubation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Pathogenic LRRK2 or knockdown of RAB8A causes accumulation of EGF in a RAB4-positive endocytic compartment. A , HeLa cells were transfected with either empty pCMV vector or pathogenic LRRK2 or cotransfected with GFP-tagged RAB4, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. B , cells were transfected as indicated followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. C , example of HeLa cells cotransfected with GFP-RAB4 and either empty pCMV vector or pathogenic LRRK2. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. Scale bar , 10 μm. D , quantification of colocalization of Alexa647-EGF with GFP-RAB4 (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 6 independent experiments. ***, p < 0.005. E , example of HeLa cells cotransfected with GFP-RAB4 and either ctrl-siRNA or RAB8A-siRNA. Live pictures were taken as described above. Arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. Scale bar , 10 μm. F , quantification of colocalization of Alexa647-EGF with GFP-RAB4 (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. All error bars represent S.E.M.

Article Snippet: Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP (1:2000; Abcam, ab6556), a mouse monoclonal anti-GAPDH (1:2000; Abcam, ab9484), a mouse monoclonal anti-LRRK2 (1:1000; NeuroMab, 75-253), a mouse monoclonal anti-RAB8A antibody (1:1000; BD Biosciences, 610844), a mouse monoclonal anti-tubulin antibody (clone DM1A; 1:10,000; Sigma), a sheep polyclonal anti-RAB8A antibody (1:500; S969D, MRC-PPU Reagents), a rabbit polyclonal anti-RAB7 antibody (1:1000; Sigma, R4779), a mouse monoclonal anti-RAB11 antibody (1:1000; BD Biosciences, 610656), and a knockout-validated rabbit monoclonal anti-RAB4 antibody (1:1000; Abcam, ab109009).

Techniques: Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Accumulation of EGF in a RAB4-positive endocytic compartment and deficits in EGFR recycling due to knockdown of RAB8A are rescued by active RAB7A expression. A , example of HeLa cells cotransfected with GFP-RAB4 and either ctrl-siRNA or RAB8A-siRNA with or without RAB7A-Q67L expression as indicated. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. An independent picture (543 HeNe laser line) was acquired to confirm coexpression of the distinct mRFP-tagged RAB7A constructs in all cases. Scale bar , 10 μm. B , quantification of colocalization of Alexa647-EGF with GFP-RAB4 and either ctrl-siRNA or RAB8A-siRNA in the presence or absence of distinct RAB7A constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01; ***, p < 0.005. C , HeLa cells were treated with ctrl-siRNA or RAB8A-siRNA as indicated and transfected with the indicated RAB7A constructs, and cell extracts (30 μg) were analyzed by Western blotting for RAB8A protein levels, mRFP-RAB7A protein levels (anti-RAB7 antibody), and GAPDH as a loading control. D , HeLa cells were treated with either ctrl-siRNA or RAB8A-siRNA as indicated with or without cotransfection with the indicated RAB7A constructs. EGFR recycling assays were performed as described under “Materials and methods,” revealing a deficit in EGFR surface levels and EGFR recycling upon RAB8A-siRNA, which was rescued upon expression of active RAB7A. n = 3 independent experiments. *, p < 0.05; ***, p < 0.005; ****, p < 0.001. A.U. , arbitrary units. All error bars represent S.E.M.

Article Snippet: Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP (1:2000; Abcam, ab6556), a mouse monoclonal anti-GAPDH (1:2000; Abcam, ab9484), a mouse monoclonal anti-LRRK2 (1:1000; NeuroMab, 75-253), a mouse monoclonal anti-RAB8A antibody (1:1000; BD Biosciences, 610844), a mouse monoclonal anti-tubulin antibody (clone DM1A; 1:10,000; Sigma), a sheep polyclonal anti-RAB8A antibody (1:500; S969D, MRC-PPU Reagents), a rabbit polyclonal anti-RAB7 antibody (1:1000; Sigma, R4779), a mouse monoclonal anti-RAB11 antibody (1:1000; BD Biosciences, 610656), and a knockout-validated rabbit monoclonal anti-RAB4 antibody (1:1000; Abcam, ab109009).

Techniques: Expressing, Construct, Transfection, Western Blot, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Expression of dominant-negative RAB7A causes defects in EGFR trafficking, accumulation of EGF in a RAB4-positive endocytic compartment, and deficits in EGFR recycling, which are reversed upon active RAB8A expression. A , HeLa cells were transfected with either empty pCMV vector ( ctrl ) or with dominant-negative RAB7A (RAB7A-T22N) in the presence or absence of active RAB8A (RAB8A-Q67L), and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. B , cells were transfected with the indicated constructs followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. C , example of HeLa cells cotransfected with GFP-RAB4 and either mRFP-RAB7A-T22N or mRFP-RAB7A-T22N and FLAG-tagged RAB8A-Q67L as indicated. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. An independent picture (543 HeNe laser line) was acquired to confirm coexpression of the mRFP-tagged RAB7A constructs in all cases. Scale bar , 10 μm. D , quantification of colocalization of Alexa647-EGF with GFP-RAB4 in the presence or absence of the distinct RAB7A constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. E , quantification of colocalization of Alexa647-EGF with GFP-RAB4 in the presence or absence of RAB7A-T22N and RAB8A-Q67L constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. *, p < 0.05. F , HeLa cells were transfected with the indicated constructs, and cell extracts (30 μg) were analyzed by Western blotting for mRFP-tagged RAB7A-T22N, FLAG-tagged RAB8A-Q67L, and GAPDH as a loading control. G , HeLa cells were transfected with either empty pCMV vector or dominant-negative RAB7A-T22N in the absence or presence of RAB8A-Q67L, and EGFR surface levels and EGFR recycling were determined at the indicated time points. n = 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005. A.U. , arbitrary units. All error bars represent S.E.M.

Article Snippet: Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP (1:2000; Abcam, ab6556), a mouse monoclonal anti-GAPDH (1:2000; Abcam, ab9484), a mouse monoclonal anti-LRRK2 (1:1000; NeuroMab, 75-253), a mouse monoclonal anti-RAB8A antibody (1:1000; BD Biosciences, 610844), a mouse monoclonal anti-tubulin antibody (clone DM1A; 1:10,000; Sigma), a sheep polyclonal anti-RAB8A antibody (1:500; S969D, MRC-PPU Reagents), a rabbit polyclonal anti-RAB7 antibody (1:1000; Sigma, R4779), a mouse monoclonal anti-RAB11 antibody (1:1000; BD Biosciences, 610656), and a knockout-validated rabbit monoclonal anti-RAB4 antibody (1:1000; Abcam, ab109009).

Techniques: Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Construct, Western Blot