australia atcc 49542 x bovienii s feltiae  (ATCC)


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    ATCC australia atcc 49542 x bovienii s feltiae
    Australia Atcc 49542 X Bovienii S Feltiae, supplied by ATCC, 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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    egfp rab3a  (Addgene inc)

     
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    Addgene inc egfp rab3a
    Plasmids used in this study.
    Egfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS–Hook–FHIP complexes"

    Article Title: Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS–Hook–FHIP complexes

    Journal: eLife

    doi: 10.7554/eLife.74538

    Plasmids used in this study.
    Figure Legend Snippet: Plasmids used in this study.

    Techniques Used:

    pegfp rab3a wt  (Addgene inc)

     
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    Addgene inc pegfp rab3a wt
    Analysis of the recruitment of Rab proteins to TcPV.
    Pegfp Rab3a Wt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Endocytic Rabs Are Recruited to the Trypanosoma cruzi Parasitophorous Vacuole and Contribute to the Process of Infection in Non-professional Phagocytic Cells"

    Article Title: Endocytic Rabs Are Recruited to the Trypanosoma cruzi Parasitophorous Vacuole and Contribute to the Process of Infection in Non-professional Phagocytic Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2020.536985

    Analysis of the recruitment of Rab proteins to TcPV.
    Figure Legend Snippet: Analysis of the recruitment of Rab proteins to TcPV.

    Techniques Used:

    pegfp rab3a  (Addgene inc)

     
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    Addgene inc pegfp rab3a
    RAW264.7 macrophages were electroporated with <t>EGFP‐Rab3A,</t> EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.
    Pegfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "LRRK 2 activation controls the repair of damaged endomembranes in macrophages"

    Article Title: LRRK 2 activation controls the repair of damaged endomembranes in macrophages

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020104494

    RAW264.7 macrophages were electroporated with EGFP‐Rab3A, EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.
    Figure Legend Snippet: RAW264.7 macrophages were electroporated with EGFP‐Rab3A, EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.

    Techniques Used: Immunofluorescence, Imaging, Western Blot, Confocal Microscopy, Fluorescence

    egfp rab3a  (Addgene inc)

     
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    Addgene inc egfp rab3a
    DRG neurons in MFCs were transfected with Halo-Na V 1.7 and either EGFP-tagged Rab6A or <t>Rab3A</t> constructs. OPAL imaging was used to visualize anterograde Halo-Na V 1.7 labeled with JF646-Halo, and two-color time-lapse imaging was performed. ( A ) Rab6A shows cotransport with Halo-Na V 1.7. Selected frames from a time-lapse movie show the position of an anterograde vesicle containing Halo-Na V 1.7 (magenta arrowheads) and EGFP-Rab6A (green arrowheads) over time. The overlay (white arrowheads) demonstrates comovement. The corresponding kymographs show a second vesicle containing both Na V 1.7 and Rab6A as demonstrated by the overlapping lines on the kymograph. ( B ) Rab3A is an example of a Rab that does not cotransport with Na V 1.7. The image sequence shows distinct Na V 1.7 (magenta arrowheads) and Rab3A (green arrowheads), demonstrating independent movement. This can be visualized by distinct magenta and green lines on the kymograph. ( C ) DRG neurons in MFCs were transfected with Na V 1.7 and either EGFP-tagged Rab1A, Rab2, Rab3A, Rab5A, Rab6A, Rab7, Rab8A, or Rab10. Anterograde Na V 1.7 was labeled with JF646-Halo only in the somatic chamber, and two-color time-lapse imaging was performed. Kymographs were created for each Rab-Na V 1.7 pair, and vesicle tracks were visually inspected and classified as Rab only, Na V 1.7 only, or both. Rab6A shows a much higher association with Na V 1.7 than any of the other Rabs investigated: Rab1A: 4%, 85 vesicles, 12 axons, 2 cultures; Rab2: 6%, 103 vesicles, 10 axons, 2 cultures; Rab3A: 7%, 115 vesicles, 8 axons, 2 cultures; Rab5A: 8%, 108 vesicles, 14 axons, 2 cultures; Rab6A: 62%, 104 vesicles, 10 axons, 4 cultures; Rab7A: 1%, 92 vesicles, 10 axons, 2 cultures; Rab8A: 13%, 83 vesicles, 10 axons, 2 cultures; Rab10: 12%, 99 vesicles, 12 axons, 2 cultures.
    Egfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Building sensory axons: Delivery and distribution of Na V 1.7 channels and effects of inflammatory mediators"

    Article Title: Building sensory axons: Delivery and distribution of Na V 1.7 channels and effects of inflammatory mediators

    Journal: Science Advances

    doi: 10.1126/sciadv.aax4755

    DRG neurons in MFCs were transfected with Halo-Na V 1.7 and either EGFP-tagged Rab6A or Rab3A constructs. OPAL imaging was used to visualize anterograde Halo-Na V 1.7 labeled with JF646-Halo, and two-color time-lapse imaging was performed. ( A ) Rab6A shows cotransport with Halo-Na V 1.7. Selected frames from a time-lapse movie show the position of an anterograde vesicle containing Halo-Na V 1.7 (magenta arrowheads) and EGFP-Rab6A (green arrowheads) over time. The overlay (white arrowheads) demonstrates comovement. The corresponding kymographs show a second vesicle containing both Na V 1.7 and Rab6A as demonstrated by the overlapping lines on the kymograph. ( B ) Rab3A is an example of a Rab that does not cotransport with Na V 1.7. The image sequence shows distinct Na V 1.7 (magenta arrowheads) and Rab3A (green arrowheads), demonstrating independent movement. This can be visualized by distinct magenta and green lines on the kymograph. ( C ) DRG neurons in MFCs were transfected with Na V 1.7 and either EGFP-tagged Rab1A, Rab2, Rab3A, Rab5A, Rab6A, Rab7, Rab8A, or Rab10. Anterograde Na V 1.7 was labeled with JF646-Halo only in the somatic chamber, and two-color time-lapse imaging was performed. Kymographs were created for each Rab-Na V 1.7 pair, and vesicle tracks were visually inspected and classified as Rab only, Na V 1.7 only, or both. Rab6A shows a much higher association with Na V 1.7 than any of the other Rabs investigated: Rab1A: 4%, 85 vesicles, 12 axons, 2 cultures; Rab2: 6%, 103 vesicles, 10 axons, 2 cultures; Rab3A: 7%, 115 vesicles, 8 axons, 2 cultures; Rab5A: 8%, 108 vesicles, 14 axons, 2 cultures; Rab6A: 62%, 104 vesicles, 10 axons, 4 cultures; Rab7A: 1%, 92 vesicles, 10 axons, 2 cultures; Rab8A: 13%, 83 vesicles, 10 axons, 2 cultures; Rab10: 12%, 99 vesicles, 12 axons, 2 cultures.
    Figure Legend Snippet: DRG neurons in MFCs were transfected with Halo-Na V 1.7 and either EGFP-tagged Rab6A or Rab3A constructs. OPAL imaging was used to visualize anterograde Halo-Na V 1.7 labeled with JF646-Halo, and two-color time-lapse imaging was performed. ( A ) Rab6A shows cotransport with Halo-Na V 1.7. Selected frames from a time-lapse movie show the position of an anterograde vesicle containing Halo-Na V 1.7 (magenta arrowheads) and EGFP-Rab6A (green arrowheads) over time. The overlay (white arrowheads) demonstrates comovement. The corresponding kymographs show a second vesicle containing both Na V 1.7 and Rab6A as demonstrated by the overlapping lines on the kymograph. ( B ) Rab3A is an example of a Rab that does not cotransport with Na V 1.7. The image sequence shows distinct Na V 1.7 (magenta arrowheads) and Rab3A (green arrowheads), demonstrating independent movement. This can be visualized by distinct magenta and green lines on the kymograph. ( C ) DRG neurons in MFCs were transfected with Na V 1.7 and either EGFP-tagged Rab1A, Rab2, Rab3A, Rab5A, Rab6A, Rab7, Rab8A, or Rab10. Anterograde Na V 1.7 was labeled with JF646-Halo only in the somatic chamber, and two-color time-lapse imaging was performed. Kymographs were created for each Rab-Na V 1.7 pair, and vesicle tracks were visually inspected and classified as Rab only, Na V 1.7 only, or both. Rab6A shows a much higher association with Na V 1.7 than any of the other Rabs investigated: Rab1A: 4%, 85 vesicles, 12 axons, 2 cultures; Rab2: 6%, 103 vesicles, 10 axons, 2 cultures; Rab3A: 7%, 115 vesicles, 8 axons, 2 cultures; Rab5A: 8%, 108 vesicles, 14 axons, 2 cultures; Rab6A: 62%, 104 vesicles, 10 axons, 4 cultures; Rab7A: 1%, 92 vesicles, 10 axons, 2 cultures; Rab8A: 13%, 83 vesicles, 10 axons, 2 cultures; Rab10: 12%, 99 vesicles, 12 axons, 2 cultures.

    Techniques Used: Transfection, Construct, Imaging, Labeling, Sequencing

    gfp rab3a  (Addgene inc)

     
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    Addgene inc gfp rab3a
    Gfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp rab3a  (Addgene inc)

     
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    Addgene inc gfp rab3a
    ( A ) Phos-tag SDS-PAGE of 14 Rabs co-expressed with different LRRK2 variants (KD = kinase dead, wt = wild type, GS= G2019S, RG = R1441G, YC= Y1699C) in HEK293 cells. Filled circles indicate unmodified Rabs and open circles, phosphorylated forms (MLi-2 = 100 nM, 1 hr). ( B ) MS-quantified <t>Rab3A-pT86,</t> Rab3B-pT86, Rab8A-pT72, Rab10-pT73 and Rab12-pS106 peptide intensities from mock and LRRK2-Y1699C transfected cells. Open circles indicate imputed values. ( C ) Western blot analysis of HA-tagged Rabs co-expressed with LRRK2-Y1699C in HEK293 cells (-/+100 nM MLi-2, 2 hr) using polyclonal anti-phospho-Rab8 and HA antibodies. ( D ) Same as ( C ) but Rabs were immunoprecipitated using the anti-pRab8 antibody and HA antibody was used for detection. ( E ) Western blot of endogenous Rabs in wild type and LRRK2-R1441C MEFs (-/+100 nM MLi-2, 1 hr) using the indicated antibodies (left) and immunoprecipitation of Rabs using anti-pRab8 antibody (right). ( F ) MS-quantified Rab phosphosite intensities of pRab8 immunoprecipitation in R1441C MEFs (-/+100 nM MLi-2, 2 hr). Error bars are mean ±SEM (n = 3). ( G ) 10 Rab proteins at endogenous expression levels are phosphorylated by LRRK2 in cells (green). Rab5A is not (red) and Rab5B, Rab5C and Rab29 are possibly phosphorylated (grey).
    Gfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis"

    Article Title: Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

    Journal: eLife

    doi: 10.7554/eLife.31012

    ( A ) Phos-tag SDS-PAGE of 14 Rabs co-expressed with different LRRK2 variants (KD = kinase dead, wt = wild type, GS= G2019S, RG = R1441G, YC= Y1699C) in HEK293 cells. Filled circles indicate unmodified Rabs and open circles, phosphorylated forms (MLi-2 = 100 nM, 1 hr). ( B ) MS-quantified Rab3A-pT86, Rab3B-pT86, Rab8A-pT72, Rab10-pT73 and Rab12-pS106 peptide intensities from mock and LRRK2-Y1699C transfected cells. Open circles indicate imputed values. ( C ) Western blot analysis of HA-tagged Rabs co-expressed with LRRK2-Y1699C in HEK293 cells (-/+100 nM MLi-2, 2 hr) using polyclonal anti-phospho-Rab8 and HA antibodies. ( D ) Same as ( C ) but Rabs were immunoprecipitated using the anti-pRab8 antibody and HA antibody was used for detection. ( E ) Western blot of endogenous Rabs in wild type and LRRK2-R1441C MEFs (-/+100 nM MLi-2, 1 hr) using the indicated antibodies (left) and immunoprecipitation of Rabs using anti-pRab8 antibody (right). ( F ) MS-quantified Rab phosphosite intensities of pRab8 immunoprecipitation in R1441C MEFs (-/+100 nM MLi-2, 2 hr). Error bars are mean ±SEM (n = 3). ( G ) 10 Rab proteins at endogenous expression levels are phosphorylated by LRRK2 in cells (green). Rab5A is not (red) and Rab5B, Rab5C and Rab29 are possibly phosphorylated (grey).
    Figure Legend Snippet: ( A ) Phos-tag SDS-PAGE of 14 Rabs co-expressed with different LRRK2 variants (KD = kinase dead, wt = wild type, GS= G2019S, RG = R1441G, YC= Y1699C) in HEK293 cells. Filled circles indicate unmodified Rabs and open circles, phosphorylated forms (MLi-2 = 100 nM, 1 hr). ( B ) MS-quantified Rab3A-pT86, Rab3B-pT86, Rab8A-pT72, Rab10-pT73 and Rab12-pS106 peptide intensities from mock and LRRK2-Y1699C transfected cells. Open circles indicate imputed values. ( C ) Western blot analysis of HA-tagged Rabs co-expressed with LRRK2-Y1699C in HEK293 cells (-/+100 nM MLi-2, 2 hr) using polyclonal anti-phospho-Rab8 and HA antibodies. ( D ) Same as ( C ) but Rabs were immunoprecipitated using the anti-pRab8 antibody and HA antibody was used for detection. ( E ) Western blot of endogenous Rabs in wild type and LRRK2-R1441C MEFs (-/+100 nM MLi-2, 1 hr) using the indicated antibodies (left) and immunoprecipitation of Rabs using anti-pRab8 antibody (right). ( F ) MS-quantified Rab phosphosite intensities of pRab8 immunoprecipitation in R1441C MEFs (-/+100 nM MLi-2, 2 hr). Error bars are mean ±SEM (n = 3). ( G ) 10 Rab proteins at endogenous expression levels are phosphorylated by LRRK2 in cells (green). Rab5A is not (red) and Rab5B, Rab5C and Rab29 are possibly phosphorylated (grey).

    Techniques Used: SDS Page, Transfection, Western Blot, Immunoprecipitation, Expressing

    australia atcc 49542 x bovienii s feltiae  (ATCC)


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    ATCC australia atcc 49542 x bovienii s feltiae
    Australia Atcc 49542 X Bovienii S Feltiae, supplied by ATCC, 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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    ATCC australia atcc 49542 x bovienii s feltiae
    Australia Atcc 49542 X Bovienii S Feltiae, supplied by ATCC, 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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    egfp rab3a  (Addgene inc)
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    Addgene inc egfp rab3a
    Plasmids used in this study.
    Egfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of the recruitment of Rab proteins to TcPV.
    Pegfp Rab3a Wt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp rab3a wt/product/Addgene inc
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    Addgene inc pegfp rab3a
    RAW264.7 macrophages were electroporated with <t>EGFP‐Rab3A,</t> EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.
    Pegfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp rab3a  (Addgene inc)
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    Addgene inc gfp rab3a
    RAW264.7 macrophages were electroporated with <t>EGFP‐Rab3A,</t> EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.
    Gfp Rab3a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Plasmids used in this study.

    Journal: eLife

    Article Title: Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS–Hook–FHIP complexes

    doi: 10.7554/eLife.74538

    Figure Lengend Snippet: Plasmids used in this study.

    Article Snippet: EGFP-Rab3A , Marci Scidmore ( ) , https://www.addgene.org/49542/.

    Techniques:

    Analysis of the recruitment of Rab proteins to TcPV.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Endocytic Rabs Are Recruited to the Trypanosoma cruzi Parasitophorous Vacuole and Contribute to the Process of Infection in Non-professional Phagocytic Cells

    doi: 10.3389/fcimb.2020.536985

    Figure Lengend Snippet: Analysis of the recruitment of Rab proteins to TcPV.

    Article Snippet: pEGFPC3 encoding GFP–VAMP3 and GFP–VAMP7 were previously characterized by Dr. Thierry Galli (The École des Neurosciences de Paris Île-de-France, Paris, France) (Martinez-Arca et al., , ) and are available at Addgene. pEGFP-Rab1 WT, pEGFP-Rab3a WT, pEGFP-Rab4 WT, pEGFP-Rab6 WT, pEGFP-Rab25 WT, pEGFP-Rab22a WT, and pEGFP-Rab22a S22N were kindly provided by Dr. Javier Magadán (Instituto de Histología y Embriología de Mendoza “Dr.

    Techniques:

    RAW264.7 macrophages were electroporated with EGFP‐Rab3A, EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: LRRK 2 activation controls the repair of damaged endomembranes in macrophages

    doi: 10.15252/embj.2020104494

    Figure Lengend Snippet: RAW264.7 macrophages were electroporated with EGFP‐Rab3A, EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.

    Article Snippet: Further plasmids used in this study are pEGFP‐Rab8A‐T22N (Addgene: 86077), pEGFP‐Rab8A‐Q67L (Addgene: 86076), pEGFP‐Rab3A (Addgene: 49542), pEGFP‐Rab10 (Addgene: 49472), pEGFP‐Rab35 (Addgene: 47424) and pTEC19 (Addgene: 30178) for transformation of M. tuberculosis .

    Techniques: Immunofluorescence, Imaging, Western Blot, Confocal Microscopy, Fluorescence