sirna for rab8a  (Thermo Fisher)

Journal: Cancers

Article Title: Controlled Plasma Membrane Delivery of FGFR1 and Modulation of Signaling by a Novel Regulated Anterograde RTK Transport Pathway

doi: 10.3390/cancers15245837

Figure Lengend Snippet: Characterization of the RART vesicles. ( A ) R1-GFP-containing vesicles do not overlap with GLUT4-positive vesicles. HEK293-R1 cells co-expressing R1-GFP and V5 epitope-tagged GLUT4 were fixed. R1-GFP was stained with FGFR1-Ab2 (detecting intracellular domain) and GLUT4 detected with anti-V5 antibody. The two molecules do not overlap. The two boxed areas are enlarged and merged in the lower panel. ( B ) HEK293-R1 cells were co-stained with anti-GFP (green) and anti-GM130 (marking Golgi; red). The R1 vesicles do not overlap significantly with GM130-positive staining. ( C ) Stability of R1-GFP vesicles was confirmed by Brefeldin-A treatment, which resulted in collapse of Golgi (arrow) but failed to disrupt R1-GFP localization pattern. ( D ) Transit through ER-Golgi was confirmed by 20 °C low-temperature Golgi-block in HEK293-R1 cells, which accumulated R1-GFP in Golgi at 20 °C (bottom right), compared to 37 °C (top right). Bright-field (with Nomarski optics) views of the same cell populations are shown on the left (Nom.). ( E ) Subcellular localizations of RED fusions of Rabs, caveolins and RalA expressed in HEK293-R1-GFP are summarized, based on representative results shown in ( F ) and in . Co-localization of R1-GFP with Rab2a, Rab6a, Rab8a, RalA and caveolins is observed. Other proteins tested did not co-localize with the R1 vesicles nor alter R1-GFP localization . Co-localization and ability to induce R1-GFP PM localization was assessed from live-cell time-lapse imaging immediately followed by re-imaging cells after brief fixation (5 min, PBS + 2% paraformaldehyde). Table in ( E ) presents summary results of RED fusions’ co-localization with R1-GFP (column 3) and their ability to induce FGFR1 PM localization (column 4) when co-expressed with R1-GFP. CCV, clathrin-coated vesicle; A/TJ, adherens/tight junction; EE, early endosome; ERGIC, ER-Golgi intermediate compartment; LE, late endosome; RE, recycling endosome; TGN, trans-Golgi network. Quantification was performed as described in “Quantification of microscopic images” of “Materials and Methods”. ( F ) Representative results of RED fusions of Rabs and caveolins expressed in HEK293-R1. The R1 vesicle localization patterns are summarized in ( E ). Boxed areas in the R1-GFP panels are enlarged as the “Zoom” insets. The same zoomed areas in “Vesical markers” and “Merge” are also shown. ( G ) Schematics and domain structure of full-length FGFR1 and the C-terminal deletion of ΔCR1. SP: signal peptide; IgI, IgII, IgIII: IgG-like domains; AB: acidic box; HB: heparin-binding domain; TM: transmembrane domain; JM: juxtamembrane domain; TK1, TK2: split tyrosine kinase domains. Representative images from five independent experiments are shown. Bars are 20 μm.

Article Snippet: Kits containing Rab8a siRNAs (Cat # SR303866) and scrambled siRNAs (Cat # SR30004) were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Staining, Blocking Assay, Imaging, Binding Assay

Journal: Cancers

Article Title: Controlled Plasma Membrane Delivery of FGFR1 and Modulation of Signaling by a Novel Regulated Anterograde RTK Transport Pathway

doi: 10.3390/cancers15245837

Figure Lengend Snippet: Regulators of FGFR1 PM localization require the receptor C-terminus. ( A ) Co-expression of R1-GFP (green) and FDPS-RED (red) in HEK293-R1 cells shows FDPS presence in ER in Nx and is largely non-overlapping with R1-GFP (top row). RED-FDPS fusion was engineered to block N-terminus ER localization motif and force cytosolic localization of FDPS. RED-FDPS expression increased PM localization of R1-GFP in Nx in HEK293-R1 cells (second row, arrow). FDPS-RED presence in cytosol is increased in 16 h SS or Hx, at which time PM translocation of R1-GFP is detectable (third and fourth rows, respectively, arrows). ( B ) Expression of indicated FGFR1 deletions (schematics on top, names in column 1) and their influence on PM localization of R1-GFP in Nx, Hx and SS are summarized in Table. Following transient expression in HEK293 parental cells, subcellular localization of FGFR1 deletions, C1-4, was studied in Nx, SS and Hx, and summary of these results are shown in column 2. Next, the deletion constructs were expressed in HEK293-R1 cells and their effects on R1-GFP localization in Nx, SS and Hx were studied, and results are shown in column 3 (RART = R1 vesicle). See G legend for domain designation. ( C ) FDPS localization to the R1 vesicle using ΔCR1-FDPS-GFP chimera results in C1-RED recruitment to the RART vesicles (top row), although no detectable PM localization of ΔCR1-FDPS-GFP was seen in Nx. C4-RED expression, which contains full FGFR1 C-terminus, induced PM localization of ΔCR1-FDPS-GFP (bottom row, arrow). ( D ) After fixation and indirect IF with RalA antibody, increased PM association of endogenous RalA was observed in SS and Hx (top row; compare middle and right panels with left panel, arrows) in HEK293-R1 cells. In cells transiently expressing constitutively active RalA-G23V (asterisks), PM localization of R1-GFP is detected in Nx (second row, arrow in inset), compared to neighboring cells that do not express RalA-G23V and show no PM localization in Nx. Conversely, cells expressing dominant-negative RalA-S28N (asterisks, third row) show inhibition of R1-GFP PM localization in Hx (third row, insets), while neighboring non-expressing cells show PM localization (sharp yellow arrows). ( E ) HEK293-R1 and HEK293-ΔCR1 cells were transfected with indicated RED fusions (column 1). Localization of Rab2a, Rab6a, Rab8a, Caveolin-1, Caveolin-2 and RalA was examined in Nx, SS and Hx and the influence of FGFR1 C-terminus was assessed. The Table summarizes semi-quantitative observations of presence (+) or absence (−) of these proteins in the R1 or the ΔCR1 vesicles. Strongest presence is indicated by ++++. Color-shaded values indicate low or no localization (yellow for the R1 data and green for the ΔR1 data). ( F ) Expression of constitutively active RED-Rab8a(Q67L) in HEK293-R1 cells stimulates PM localization of R1-GFP in Nx (arrow). Quantification in ( B ) was performed as described in . Quantitation in ( E ) was performed as described in . Yellow boxes are regions of interest that are enlarged (Zoom). Bars are 20 μm.

Article Snippet: Kits containing Rab8a siRNAs (Cat # SR303866) and scrambled siRNAs (Cat # SR30004) were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Blocking Assay, Translocation Assay, Construct, Dominant Negative Mutation, Inhibition, Transfection, Quantitation Assay

Journal: Cancers

Article Title: Controlled Plasma Membrane Delivery of FGFR1 and Modulation of Signaling by a Novel Regulated Anterograde RTK Transport Pathway

doi: 10.3390/cancers15245837

Figure Lengend Snippet: Identification of regulatory ON/OFF switches of the RART pathway. The schematics on left and right of image panels show ON/OFF switches of the RART pathway. Note: since RED is fused to C-terminus of Rab proteins, their recruitment to the R1 vesicle is CaaX-independent and is dependent on localization as ΔCR1 fusions. ( A ) The inhibitory effect of Rab2a on PM localization of R1-GFP was studied using ΔCR1-Rab2a-RED fusion. The top row shows that in the absence of FGFR1 C-terminus in ΔCR1-GFP, Rab2a is not released from the R1 vesicles in 24 h SS. In second row, cells expressing ΔCR1-Rab2a-RED (asterisks) show no PM translocation of R1-GFP in 24 h Hx (dashed white arrow), while neighboring cells show R1-GFP on the PM (yellow arrow). The third row shows that Rab2a is released in Nx upon direct recruitment of FDPS to the R1 vesicle as ΔCR1-FDPS-GFP chimera. ( B ) Release of Rab2a using ΔCR1-FDPS-GFP failed to promote PM translocation even when Rab8a level was increased by co-expressing RED-Rab8a (top row). In the bottom row, direct recruitment of constitutively active Rab8a to the R1 vesicle in cells expressing ΔCR1-Rab8a(Q67L)-RED (asterisks), led to presence of ΔCR1-GFP on the PM (yellow arrow in inset), while neighboring untransfected cells show no PM localization in Nx (dashed white arrow), showing that Rab8a activation on the R1 vesicle is necessary to stimulate PM translocation. ( C ) RED-Rab6a is present in the R1 vesicles (top row), but not in the ΔCR1 vesicles (second row). Release of inhibitory Rab2a using ΔCR1-FDPS-GFP failed to recruit RED-Rab6a to the ΔCR1 vesicles (third row). Three insets are shown in enlarged views. Vesicle localization of Rab6a using ΔCR1-Rab6a-RED (inset 1 in the bottom row), accompanied by release of Rab2a using ΔCR1-FDPS-GFP, led to detectable increase in PM localization of ΔCR1-FDPS-GFP (bottom row, arrows in insets 2 and 3). ( D ) The model for regulatory ON/OFF switches in the RART pathway and their activation sequence is presented. Yellow boxes are regions of interest that are enlarged (Zoom). Bars are 20 μm.

Article Snippet: Kits containing Rab8a siRNAs (Cat # SR303866) and scrambled siRNAs (Cat # SR30004) were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Translocation Assay, Activation Assay, Sequencing

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

Techniques: Knockdown, Transfection, Western Blot, Control, 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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

Techniques: Knockdown, 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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

Techniques: Knockdown, Expressing, Construct, Transfection, Western Blot, Control, 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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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: An siRNA-resistant form of RAB8A ( ) was generated by introducing three silent mutations into the target sequence of the seed region of the RAB8A-siRNA (Ambion, Thermo Fisher, ID s8679, catalog number 4390824).

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

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Primer sequences to clone hSVCT1 and Rab8a and real-time PCR primers

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Real-time Polymerase Chain Reaction

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2, HT-29, NCM460 and HuTU-80 cells were transiently co-transfected with hSVCT1-YEP and DsRed-Rab8a constructs. Data are from n > 6-10 transfected cells. Scale bar is 10 μm

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Transfection, Construct

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) 14C-AA uptake (32 μM) was performed on Rab8a siRNA treated hSVCT1 expressing Caco-2 cells. Data are mean ± SE of at least three separate experiments performed on different batches of cells on separate occasions. *p < 0.02. B) Real-time PCR was performed using gene specific primers for Rab8a and β-actin from total RNA isolated from Rab8a siRNA and control siRNA (scrambled) treated Caco-2 cells. Data are mean ± SE of at least three independent experiments. *p < 0.01. C, Top, Western blot analysis was performed on cell extract (60 μg) isolated from control (left) and Rab8a siRNAs treated Caco-2 cells (right). Blots were incubated with rabbit polyclonal anti-human Rab8a specific antibodies (top) along with β-actin antibodies (bottom). Bottom, densitometric quantification of the immunoreactive bands. Data are mean ± SE of at least three separate sample preparations. *p < 0.01. D) Carrier-mediated 14C-AA uptake (32 μM) by Rab8a KO mice jejunal portion was performed as described in “Materials and Methods”. Values are mean ± SE of at least three separate uptake determinations from multiple sets of mice. *p < 0.01. E) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type (litter-mate) mice jejunal mucosa using mouse Rab8a and β-actin gene-specific primers. Data are mean ± SE of at least three separate samples from three different mice. *p < 0.01. F, Top) Western blot analysis was performed on Rab8a KO (right) and wild-type litter-mate (left) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit polyclonal anti-mouse Rab8a antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three sets of samples from three different mice.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) Real-time PCR was performed using primers for hSVCT1, hSVCT2 and β-actin on total RNA isolated from control (scrambled) and Rab8a siRNAs treated Caco-2 cells. Data are mean ± SE of multiple experiments performed on different batches of cells. B, Top, western blot analysis was performed on cell extract (60 μg) isolated from control and Rab8a siRNAs treated Caco-2 cells. Blots were incubated with rabbit anti-human hSVCT1 specific antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of multiple experiments performed on separately isolated samples.*p < 0.01. C) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type litter-mate mice jejunal mucosa using mouse SVCT1, SVCT2 and β-actin primers. Data are mean ± SE of at least three separate samples from three different mice. D, Top, western blot analysis was performed on Rab8a KO and wild-type (litter-mate) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit anti-mouse SVCT1 antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three separate samples from three mice. *p < 0.02.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2 cells were transiently transfected with Rab8a siRNA or control siRNA. Forty eight hours after transfection cells were processed for biotinylation. Equal amount of protein was loaded onto pre-made 4-12% mini-gel and western blot was performed using anti-hSVCT1 antibody. The level of cell surface expression was normalized relative to the total amount of cellular hSVCT1 protein. Densitometric values are from mean ± SE of four independent experiments. Inset shows representative western blot images. *p < 0.01.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Transfection, Control, Western Blot, Expressing

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Hu-Tu-80 cells were co-transfected with hSVCT1-YEP, LAMP1-RFP, control or Rab8a siRNAs. Live cell confocal imaging (laterl sections-xy) was performed after 48 h of transfection. Data are from n > 6-10 transfected cells and representative images were shown. Scale bar is 10 μm.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Transfection, Control, Imaging