Structured Review

Millipore anti rab14 c terminal antibody
Mutation of serines 580 and 582 does not affect the localization of RCP with endogenous Rab11 or <t>Rab14.</t> HeLa cells expressing the indicated GFP-RCP constructs were fixed and immunolabeled with antibodies that recognize endogenous Rab11 ( A ) and Rab14 (
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Images

1) Product Images from "Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *"

Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.612366

Mutation of serines 580 and 582 does not affect the localization of RCP with endogenous Rab11 or Rab14. HeLa cells expressing the indicated GFP-RCP constructs were fixed and immunolabeled with antibodies that recognize endogenous Rab11 ( A ) and Rab14 (
Figure Legend Snippet: Mutation of serines 580 and 582 does not affect the localization of RCP with endogenous Rab11 or Rab14. HeLa cells expressing the indicated GFP-RCP constructs were fixed and immunolabeled with antibodies that recognize endogenous Rab11 ( A ) and Rab14 (

Techniques Used: Mutagenesis, Expressing, Construct, Immunolabeling

RCP displays greater colocalization with Rab11. A , HeLa cells were fixed and co-labeled with antibodies that recognize endogenous RCP and Rab11 or Rab14. B, quantitative analysis of the colocalization coefficients of endogenous RCP with endogenous Rab11
Figure Legend Snippet: RCP displays greater colocalization with Rab11. A , HeLa cells were fixed and co-labeled with antibodies that recognize endogenous RCP and Rab11 or Rab14. B, quantitative analysis of the colocalization coefficients of endogenous RCP with endogenous Rab11

Techniques Used: Labeling

Rab14 interacts with the RBD of RCP. A, schematic of RCP indicating conserved domains and the location of mutated residues. B, ClustalW alignment of the C-terminal region of the class I FIPs. C, far Western protein-protein interaction assays. Equal amounts
Figure Legend Snippet: Rab14 interacts with the RBD of RCP. A, schematic of RCP indicating conserved domains and the location of mutated residues. B, ClustalW alignment of the C-terminal region of the class I FIPs. C, far Western protein-protein interaction assays. Equal amounts

Techniques Used: Western Blot

Isothermal titration calorimetry of Rab14/effector interactions. A, raw data ( top panel ) and integrated heats ( bottom panel ) of wtRCP(581–649) (600 μ m ) injected into 30 μ m Rab14(2–175). B, injection of 600 μ m Rab14(2–175)
Figure Legend Snippet: Isothermal titration calorimetry of Rab14/effector interactions. A, raw data ( top panel ) and integrated heats ( bottom panel ) of wtRCP(581–649) (600 μ m ) injected into 30 μ m Rab14(2–175). B, injection of 600 μ m Rab14(2–175)

Techniques Used: Isothermal Titration Calorimetry, Injection

Rab11 recruits RCP to membranes. A, HeLa cells were transfected with control siRNA (siFLuc) or siRNA-duplexes targeting Rab11 and Rab14 for 72 h. The cells were transfected with GFP-RCP WT for the final 24 h and fixed. Single 0.4-μm sections were
Figure Legend Snippet: Rab11 recruits RCP to membranes. A, HeLa cells were transfected with control siRNA (siFLuc) or siRNA-duplexes targeting Rab11 and Rab14 for 72 h. The cells were transfected with GFP-RCP WT for the final 24 h and fixed. Single 0.4-μm sections were

Techniques Used: Transfection

Structure of the Rab14-RCP complex. A, ribbon model of Rab14 ( gray ) in complex with RCP homodimer ( yellow and orange ). The switch regions are indicated in red and blue ; GTP is a green stick model, and Mg 2+ is shown as a sphere. B , superposition of Rab14
Figure Legend Snippet: Structure of the Rab14-RCP complex. A, ribbon model of Rab14 ( gray ) in complex with RCP homodimer ( yellow and orange ). The switch regions are indicated in red and blue ; GTP is a green stick model, and Mg 2+ is shown as a sphere. B , superposition of Rab14

Techniques Used:

RCP and Rab14 play a role in neuritogenesis in mouse N2a neuroblastoma cells. A, N2a cells were transfected with the indicated GFP fusion constructs ( green ) for 24 h. Cells were then incubated for a further 24 h in serum-free medium to induce cell differentiation,
Figure Legend Snippet: RCP and Rab14 play a role in neuritogenesis in mouse N2a neuroblastoma cells. A, N2a cells were transfected with the indicated GFP fusion constructs ( green ) for 24 h. Cells were then incubated for a further 24 h in serum-free medium to induce cell differentiation,

Techniques Used: Transfection, Construct, Incubation, Cell Differentiation

2) Product Images from "Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis"

Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

Journal: bioRxiv

doi: 10.1101/2020.04.21.052449

Rab14 knock-down increases time required for cells to undergo abscission. (A) Individual images taken from time-lapse analysis of dividing Rab14-KO cell (also see Supplemental Movie 3). (B) Control, Rab14-KD and Rab11a/b-KD cells were fixed and stained with anti-tubulin antibodies to identify cells in telophase. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control, Rab14-KD, Rab11a/b-KD, Rab14/11-DKD and Rab14-KO cells were analysed by time-lapse microscopy to measure the time needed to complete division (starting from metaphase). Where indicated, cells were transfected with GFP-Rab11a or GFP-Rab14. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Control or Rab14-KO cells were incubated in the absence or presence of 4 nM Latrunculin A and multi-nucleated cells counted to assess their ability to complete cytokinesis. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (F-G) Control or Rab14-KO HeLa cells were fixed and stained with phalloidin-596 and anti-tubulin antibodies. The fluorescence of phalloidin-596 at the ICB was then evaluated. The quantification is shown at panel (F). Data shown are the means and standard deviations derived from three independent experiments.
Figure Legend Snippet: Rab14 knock-down increases time required for cells to undergo abscission. (A) Individual images taken from time-lapse analysis of dividing Rab14-KO cell (also see Supplemental Movie 3). (B) Control, Rab14-KD and Rab11a/b-KD cells were fixed and stained with anti-tubulin antibodies to identify cells in telophase. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control, Rab14-KD, Rab11a/b-KD, Rab14/11-DKD and Rab14-KO cells were analysed by time-lapse microscopy to measure the time needed to complete division (starting from metaphase). Where indicated, cells were transfected with GFP-Rab11a or GFP-Rab14. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Control or Rab14-KO cells were incubated in the absence or presence of 4 nM Latrunculin A and multi-nucleated cells counted to assess their ability to complete cytokinesis. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (F-G) Control or Rab14-KO HeLa cells were fixed and stained with phalloidin-596 and anti-tubulin antibodies. The fluorescence of phalloidin-596 at the ICB was then evaluated. The quantification is shown at panel (F). Data shown are the means and standard deviations derived from three independent experiments.

Techniques Used: Staining, Derivative Assay, Time-lapse Microscopy, Transfection, Incubation, Fluorescence

Schematic representation of the proposed function for Rab14/MACF2 complex. MACF2 is recruited to the central spindle microtubules either by directly binding to microtubule bundles or by co-binding to CAMSAP3, the protein that interacts with microtubule minus-ends. MACF2 then interacts with Rab14, thus, recruiting Rab14-containing early endosomes to the central spindle. Rab11/Fip3 recycling endosomes then buds from early endosomes and is delivered to the abscission site via kinesin-2 molecular motor.
Figure Legend Snippet: Schematic representation of the proposed function for Rab14/MACF2 complex. MACF2 is recruited to the central spindle microtubules either by directly binding to microtubule bundles or by co-binding to CAMSAP3, the protein that interacts with microtubule minus-ends. MACF2 then interacts with Rab14, thus, recruiting Rab14-containing early endosomes to the central spindle. Rab11/Fip3 recycling endosomes then buds from early endosomes and is delivered to the abscission site via kinesin-2 molecular motor.

Techniques Used: Binding Assay

Rab14 binds to MACF2/CAMSAP3 complex. (A) Putative Rab14-interacting proteins identified in FLAG-Rab14 immunoprecipitation and proteomic analysis. (B) Lysates from HeLa cells expressing endogenously labelled GFP-Rab14 were incubated with either glutathione beads coated with GST or GST-anti-GFP-nanobody. The beads were then washed and bound protein analysed by western blotting. (C-D) HeLa cells transfected with Halo-MACF2 were fixed and stained with Halo ligand and anti-tubulin antibodies. (E-F) Control (E) or Rab14-KO (F) HeLa cells were transfected with GFP-CAMSAP3, then fixed and stained with anti-tubulin antibodies.
Figure Legend Snippet: Rab14 binds to MACF2/CAMSAP3 complex. (A) Putative Rab14-interacting proteins identified in FLAG-Rab14 immunoprecipitation and proteomic analysis. (B) Lysates from HeLa cells expressing endogenously labelled GFP-Rab14 were incubated with either glutathione beads coated with GST or GST-anti-GFP-nanobody. The beads were then washed and bound protein analysed by western blotting. (C-D) HeLa cells transfected with Halo-MACF2 were fixed and stained with Halo ligand and anti-tubulin antibodies. (E-F) Control (E) or Rab14-KO (F) HeLa cells were transfected with GFP-CAMSAP3, then fixed and stained with anti-tubulin antibodies.

Techniques Used: Immunoprecipitation, Expressing, Incubation, Western Blot, Transfection, Staining

Rab14 is enriched at early endosomes located at the minus-ends of central spindle microtubules. (A-C) HeLa cells expressing GFP-tagged endogenous Rab14 were fixed and stained with anti-tubulin (A), anti-EEA1 (B) or anti-Fip1 (C) antibodies. Asterisks mark the ICB. (D-E) HeLa cells in telophase were fixed and stained with anti-Rab14 (D and E), anti-tubulin (D) or anti-Fip1 (E) antibodies. (E) Schematic representation of proposed endocytic Rab14 localization. (F) HeLa cells in interphase were fixed and stained with anti-Fip1 and anti-Rab14 antibodies.
Figure Legend Snippet: Rab14 is enriched at early endosomes located at the minus-ends of central spindle microtubules. (A-C) HeLa cells expressing GFP-tagged endogenous Rab14 were fixed and stained with anti-tubulin (A), anti-EEA1 (B) or anti-Fip1 (C) antibodies. Asterisks mark the ICB. (D-E) HeLa cells in telophase were fixed and stained with anti-Rab14 (D and E), anti-tubulin (D) or anti-Fip1 (E) antibodies. (E) Schematic representation of proposed endocytic Rab14 localization. (F) HeLa cells in interphase were fixed and stained with anti-Fip1 and anti-Rab14 antibodies.

Techniques Used: Expressing, Staining

Rab14 is required for targeting Rab11-endosomes to the central spindle microtubules. (A-B) Control or Rab14-KO HeLa cells were fixed and stained with anti-tubulin and anti-Fip1 antibodies. Line-scan analysis of Fip1 fluorescence was then performed in telophase cells (see line in panel A). Asterisks mark the ICB. Data shown in (B) are the means and standard deviations derived from line-scans of four different cells. (C-D) Time-lapse images of dividing control or Rab14-KO cells expressing GFP-Fip3. Asterisks mark the ICB.
Figure Legend Snippet: Rab14 is required for targeting Rab11-endosomes to the central spindle microtubules. (A-B) Control or Rab14-KO HeLa cells were fixed and stained with anti-tubulin and anti-Fip1 antibodies. Line-scan analysis of Fip1 fluorescence was then performed in telophase cells (see line in panel A). Asterisks mark the ICB. Data shown in (B) are the means and standard deviations derived from line-scans of four different cells. (C-D) Time-lapse images of dividing control or Rab14-KO cells expressing GFP-Fip3. Asterisks mark the ICB.

Techniques Used: Staining, Fluorescence, Derivative Assay, Expressing

Rab14 is required for completion of cytokinesis. (A) Endocytic Rab GTPases present in post-mitotic MB proteome. (B) HeLa cells were transfected with dominant-negative mutants of various MB-associated Rabs. Multi-nucleated cells were then counted and expressed as percentage of total number of cells. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control and Rab14-KD cells were transfected with various GFP-tagged Rabs. The ability of overexpression to decrease multi-nucleation was then analysed. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Individual images taken from time-lapse analysis of dividing control (top row) and Rab14-KO (bottom row) cells (also see Movies 1 2).
Figure Legend Snippet: Rab14 is required for completion of cytokinesis. (A) Endocytic Rab GTPases present in post-mitotic MB proteome. (B) HeLa cells were transfected with dominant-negative mutants of various MB-associated Rabs. Multi-nucleated cells were then counted and expressed as percentage of total number of cells. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control and Rab14-KD cells were transfected with various GFP-tagged Rabs. The ability of overexpression to decrease multi-nucleation was then analysed. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Individual images taken from time-lapse analysis of dividing control (top row) and Rab14-KO (bottom row) cells (also see Movies 1 2).

Techniques Used: Transfection, Dominant Negative Mutation, Derivative Assay, Over Expression

3) Product Images from "Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis"

Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

Journal: bioRxiv

doi: 10.1101/2020.04.21.052449

Rab14 knock-down increases time required for cells to undergo abscission. (A) Individual images taken from time-lapse analysis of dividing Rab14-KO cell (also see Supplemental Movie 3). (B) Control, Rab14-KD and Rab11a/b-KD cells were fixed and stained with anti-tubulin antibodies to identify cells in telophase. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control, Rab14-KD, Rab11a/b-KD, Rab14/11-DKD and Rab14-KO cells were analysed by time-lapse microscopy to measure the time needed to complete division (starting from metaphase). Where indicated, cells were transfected with GFP-Rab11a or GFP-Rab14. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Control or Rab14-KO cells were incubated in the absence or presence of 4 nM Latrunculin A and multi-nucleated cells counted to assess their ability to complete cytokinesis. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (F-G) Control or Rab14-KO HeLa cells were fixed and stained with phalloidin-596 and anti-tubulin antibodies. The fluorescence of phalloidin-596 at the ICB was then evaluated. The quantification is shown at panel (F). Data shown are the means and standard deviations derived from three independent experiments.
Figure Legend Snippet: Rab14 knock-down increases time required for cells to undergo abscission. (A) Individual images taken from time-lapse analysis of dividing Rab14-KO cell (also see Supplemental Movie 3). (B) Control, Rab14-KD and Rab11a/b-KD cells were fixed and stained with anti-tubulin antibodies to identify cells in telophase. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control, Rab14-KD, Rab11a/b-KD, Rab14/11-DKD and Rab14-KO cells were analysed by time-lapse microscopy to measure the time needed to complete division (starting from metaphase). Where indicated, cells were transfected with GFP-Rab11a or GFP-Rab14. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Control or Rab14-KO cells were incubated in the absence or presence of 4 nM Latrunculin A and multi-nucleated cells counted to assess their ability to complete cytokinesis. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (F-G) Control or Rab14-KO HeLa cells were fixed and stained with phalloidin-596 and anti-tubulin antibodies. The fluorescence of phalloidin-596 at the ICB was then evaluated. The quantification is shown at panel (F). Data shown are the means and standard deviations derived from three independent experiments.

Techniques Used: Staining, Derivative Assay, Time-lapse Microscopy, Transfection, Incubation, Fluorescence

Schematic representation of the proposed function for Rab14/MACF2 complex. MACF2 is recruited to the central spindle microtubules either by directly binding to microtubule bundles or by co-binding to CAMSAP3, the protein that interacts with microtubule minus-ends. MACF2 then interacts with Rab14, thus, recruiting Rab14-containing early endosomes to the central spindle. Rab11/Fip3 recycling endosomes then buds from early endosomes and is delivered to the abscission site via kinesin-2 molecular motor.
Figure Legend Snippet: Schematic representation of the proposed function for Rab14/MACF2 complex. MACF2 is recruited to the central spindle microtubules either by directly binding to microtubule bundles or by co-binding to CAMSAP3, the protein that interacts with microtubule minus-ends. MACF2 then interacts with Rab14, thus, recruiting Rab14-containing early endosomes to the central spindle. Rab11/Fip3 recycling endosomes then buds from early endosomes and is delivered to the abscission site via kinesin-2 molecular motor.

Techniques Used: Binding Assay

Rab14 binds to MACF2/CAMSAP3 complex. (A) Putative Rab14-interacting proteins identified in FLAG-Rab14 immunoprecipitation and proteomic analysis. (B) Lysates from HeLa cells expressing endogenously labelled GFP-Rab14 were incubated with either glutathione beads coated with GST or GST-anti-GFP-nanobody. The beads were then washed and bound protein analysed by western blotting. (C-D) HeLa cells transfected with Halo-MACF2 were fixed and stained with Halo ligand and anti-tubulin antibodies. (E-F) Control (E) or Rab14-KO (F) HeLa cells were transfected with GFP-CAMSAP3, then fixed and stained with anti-tubulin antibodies.
Figure Legend Snippet: Rab14 binds to MACF2/CAMSAP3 complex. (A) Putative Rab14-interacting proteins identified in FLAG-Rab14 immunoprecipitation and proteomic analysis. (B) Lysates from HeLa cells expressing endogenously labelled GFP-Rab14 were incubated with either glutathione beads coated with GST or GST-anti-GFP-nanobody. The beads were then washed and bound protein analysed by western blotting. (C-D) HeLa cells transfected with Halo-MACF2 were fixed and stained with Halo ligand and anti-tubulin antibodies. (E-F) Control (E) or Rab14-KO (F) HeLa cells were transfected with GFP-CAMSAP3, then fixed and stained with anti-tubulin antibodies.

Techniques Used: Immunoprecipitation, Expressing, Incubation, Western Blot, Transfection, Staining

Rab14 is enriched at early endosomes located at the minus-ends of central spindle microtubules. (A-C) HeLa cells expressing GFP-tagged endogenous Rab14 were fixed and stained with anti-tubulin (A), anti-EEA1 (B) or anti-Fip1 (C) antibodies. Asterisks mark the ICB. (D-E) HeLa cells in telophase were fixed and stained with anti-Rab14 (D and E), anti-tubulin (D) or anti-Fip1 (E) antibodies. (E) Schematic representation of proposed endocytic Rab14 localization. (F) HeLa cells in interphase were fixed and stained with anti-Fip1 and anti-Rab14 antibodies.
Figure Legend Snippet: Rab14 is enriched at early endosomes located at the minus-ends of central spindle microtubules. (A-C) HeLa cells expressing GFP-tagged endogenous Rab14 were fixed and stained with anti-tubulin (A), anti-EEA1 (B) or anti-Fip1 (C) antibodies. Asterisks mark the ICB. (D-E) HeLa cells in telophase were fixed and stained with anti-Rab14 (D and E), anti-tubulin (D) or anti-Fip1 (E) antibodies. (E) Schematic representation of proposed endocytic Rab14 localization. (F) HeLa cells in interphase were fixed and stained with anti-Fip1 and anti-Rab14 antibodies.

Techniques Used: Expressing, Staining

Rab14 is required for targeting Rab11-endosomes to the central spindle microtubules. (A-B) Control or Rab14-KO HeLa cells were fixed and stained with anti-tubulin and anti-Fip1 antibodies. Line-scan analysis of Fip1 fluorescence was then performed in telophase cells (see line in panel A). Asterisks mark the ICB. Data shown in (B) are the means and standard deviations derived from line-scans of four different cells. (C-D) Time-lapse images of dividing control or Rab14-KO cells expressing GFP-Fip3. Asterisks mark the ICB.
Figure Legend Snippet: Rab14 is required for targeting Rab11-endosomes to the central spindle microtubules. (A-B) Control or Rab14-KO HeLa cells were fixed and stained with anti-tubulin and anti-Fip1 antibodies. Line-scan analysis of Fip1 fluorescence was then performed in telophase cells (see line in panel A). Asterisks mark the ICB. Data shown in (B) are the means and standard deviations derived from line-scans of four different cells. (C-D) Time-lapse images of dividing control or Rab14-KO cells expressing GFP-Fip3. Asterisks mark the ICB.

Techniques Used: Staining, Fluorescence, Derivative Assay, Expressing

Rab14 is required for completion of cytokinesis. (A) Endocytic Rab GTPases present in post-mitotic MB proteome. (B) HeLa cells were transfected with dominant-negative mutants of various MB-associated Rabs. Multi-nucleated cells were then counted and expressed as percentage of total number of cells. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control and Rab14-KD cells were transfected with various GFP-tagged Rabs. The ability of overexpression to decrease multi-nucleation was then analysed. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Individual images taken from time-lapse analysis of dividing control (top row) and Rab14-KO (bottom row) cells (also see Movies 1 2).
Figure Legend Snippet: Rab14 is required for completion of cytokinesis. (A) Endocytic Rab GTPases present in post-mitotic MB proteome. (B) HeLa cells were transfected with dominant-negative mutants of various MB-associated Rabs. Multi-nucleated cells were then counted and expressed as percentage of total number of cells. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control and Rab14-KD cells were transfected with various GFP-tagged Rabs. The ability of overexpression to decrease multi-nucleation was then analysed. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Individual images taken from time-lapse analysis of dividing control (top row) and Rab14-KO (bottom row) cells (also see Movies 1 2).

Techniques Used: Transfection, Dominant Negative Mutation, Derivative Assay, Over Expression

4) Product Images from "SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors"

Article Title: SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors

Journal: Nature Communications

doi: 10.1038/s41467-019-09617-9

Vps13B specifically binds to Rab14 and Rab6 and to PtdIns(3)P. a , b Association of endosomal Rab proteins with Vps13B, revealed by co-immunoprecipitation of endogenous Vps13B with GFP (or mRFP)-tagged Rab proteins. a GFP (or mRFP)-tagged proteins were precipitated from cell lysates using a GFP-trap or anti-mRFP antibody. Bound proteins were analyzed by immunoblotting for Vps13B, GFP, and mRFP. b Cross-linked cells were lysed and immunoprecipitated with anti-Vps13B antibody. The interaction of Rab proteins was detected and indicated Rab protein antibodies. c Coimmunoprecipitation of VPS13B with mRFP-Rab14-WT, GTP-locked mRFP-Rab14Q70L (QL), or GDP-locked mRFP-Rab14S25N (SN) expressed in HeLa cells. Anti-RFP antibody was used for immunoprecipitation of mRFP-tagged Rab14 proteins. There was no significant difference in the binding of Vps13B to the Rab14 variants. d Vps13B co-localizes with Rab6 and Rab14 upon expression in HeLa cells. Top panel: cells were transfected with mRFP-Rab14 and Vps13B-GFP and analyzed for the presence of GFP and mRFP, respectively, by fluorescence microscopy. Bottom panel: cells were transfected with GFP-Rab6. In this case, the distribution of endogenous Vps13B was monitored by immunocytochemistry. White arrows in the upper inserts show vesicles positive for both Vps13B and Rab14. Bottom panels show line scans on the lines in the upper panels and the percentage of colocalization of Rab14 with Vps13B. Scale bar, 10 µm. e PIP strip-binding assay of Vps13B. Perinuclear supernatant from Vps13B-GFP overexpressing cells was incubated with a membrane strip containing the phosphoinositides as indicated and probed for bound Vps13B using an anti-GFP antibody. f Binding of Rhodamine-labeled liposomes to Vps13B-GFP that was immobilized on magnetic Dynabeads. After incubation, the beads were washed, and bound liposomes were detected by Rhodamine fluorescence. Binding was only detectable when Vps13B-GFP was bound to the beads and when the liposomes contained 5% PtdIns(3)P. Error bars indicate S.E.M., *** P
Figure Legend Snippet: Vps13B specifically binds to Rab14 and Rab6 and to PtdIns(3)P. a , b Association of endosomal Rab proteins with Vps13B, revealed by co-immunoprecipitation of endogenous Vps13B with GFP (or mRFP)-tagged Rab proteins. a GFP (or mRFP)-tagged proteins were precipitated from cell lysates using a GFP-trap or anti-mRFP antibody. Bound proteins were analyzed by immunoblotting for Vps13B, GFP, and mRFP. b Cross-linked cells were lysed and immunoprecipitated with anti-Vps13B antibody. The interaction of Rab proteins was detected and indicated Rab protein antibodies. c Coimmunoprecipitation of VPS13B with mRFP-Rab14-WT, GTP-locked mRFP-Rab14Q70L (QL), or GDP-locked mRFP-Rab14S25N (SN) expressed in HeLa cells. Anti-RFP antibody was used for immunoprecipitation of mRFP-tagged Rab14 proteins. There was no significant difference in the binding of Vps13B to the Rab14 variants. d Vps13B co-localizes with Rab6 and Rab14 upon expression in HeLa cells. Top panel: cells were transfected with mRFP-Rab14 and Vps13B-GFP and analyzed for the presence of GFP and mRFP, respectively, by fluorescence microscopy. Bottom panel: cells were transfected with GFP-Rab6. In this case, the distribution of endogenous Vps13B was monitored by immunocytochemistry. White arrows in the upper inserts show vesicles positive for both Vps13B and Rab14. Bottom panels show line scans on the lines in the upper panels and the percentage of colocalization of Rab14 with Vps13B. Scale bar, 10 µm. e PIP strip-binding assay of Vps13B. Perinuclear supernatant from Vps13B-GFP overexpressing cells was incubated with a membrane strip containing the phosphoinositides as indicated and probed for bound Vps13B using an anti-GFP antibody. f Binding of Rhodamine-labeled liposomes to Vps13B-GFP that was immobilized on magnetic Dynabeads. After incubation, the beads were washed, and bound liposomes were detected by Rhodamine fluorescence. Binding was only detectable when Vps13B-GFP was bound to the beads and when the liposomes contained 5% PtdIns(3)P. Error bars indicate S.E.M., *** P

Techniques Used: Immunoprecipitation, Binding Assay, Expressing, Transfection, Fluorescence, Microscopy, Immunocytochemistry, Stripping Membranes, Incubation, Labeling

5) Product Images from "Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line"

Article Title: Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line

Journal: Virulence

doi: 10.1080/21505594.2017.1361089

Rab 14, PI3K-Akt axis and intracellular survival of S.aureus . (a) Absence of colocalization of S.aureus and Rab14 over a time course (images were taken 1.5 h post infection). Cells were infected with GFP- S.aureus . Rab14 was stained with rabbit anti-Rab14 and Rhodamine Red-conjugated donkey anti-rabbit (red) antibodies. Images are representative of triplicate coverslips in 3 independent experiments. (b) Quantification of intracellular bacteria in transfected MH-S with plasmid pcDNA3 or with Rab14 dominant negative construct (DN Rab 14) over a time course. Cells were transfected 24 h before the experiment, and cells were infected as described above. Data shown as c.f.u./well are the average of 3 independent experiments; *, p
Figure Legend Snippet: Rab 14, PI3K-Akt axis and intracellular survival of S.aureus . (a) Absence of colocalization of S.aureus and Rab14 over a time course (images were taken 1.5 h post infection). Cells were infected with GFP- S.aureus . Rab14 was stained with rabbit anti-Rab14 and Rhodamine Red-conjugated donkey anti-rabbit (red) antibodies. Images are representative of triplicate coverslips in 3 independent experiments. (b) Quantification of intracellular bacteria in transfected MH-S with plasmid pcDNA3 or with Rab14 dominant negative construct (DN Rab 14) over a time course. Cells were transfected 24 h before the experiment, and cells were infected as described above. Data shown as c.f.u./well are the average of 3 independent experiments; *, p

Techniques Used: Infection, Staining, Transfection, Plasmid Preparation, Dominant Negative Mutation, Construct

6) Product Images from "Targeting CPT1A-mediated fatty acid oxidation sensitizes nasopharyngeal carcinoma to radiation therapy"

Article Title: Targeting CPT1A-mediated fatty acid oxidation sensitizes nasopharyngeal carcinoma to radiation therapy

Journal: Theranostics

doi: 10.7150/thno.21451

The binding of CPT1A to Rab14 is enhanced in radiation-resistant NPC cells, which might promote the contact of lipid droplet and mitochondria in these cells. (A) Immunoprecipitation analysis showing the binding of CPT1A and Rab14 in CNE2-IR and HK1-IR compared with parental cells. IgG served as a negative control. (B) Immunoprecipitation analysis showing the binding of CPT1A and Rab14 in CNE2 and HK1 cells stably expressing CPT1A-V5 or empty vector. IgG served as a negative control. (C) Proximity ligation assay indicating the interaction of CPT1A and Rab14 in CNE2-IR and HK1-IR compared with parental cells at 24 h after exposure to 4 Gy IR (red: PLA positive signal; blue: DAPI, scale bar = 25 μm). (D) Based on the prediction of Fd-DCA, the N terminal of CPT1A was found to bind with the identified Site 2 of Rab14 ( left ) and the predicted top 20 strongest coupled interacting residue pairs (interacting residue contacts) of these two proteins (orange stick bonds, right ). (E) Immunoblot analysis showing translocation of CPT1A to mitochondria in CNE2 and CNE2-IR cells transfected with a Rab14 siRNA pool or negative siRNA at 24 h after exposure to 4 Gy IR. β-Actin was used as a control to confirm equal loading of protein and Hsp60 served as a control to confirm equal loading of mitochondrial fractions. (F) Confocal microscopy analysis of the co-localization of Rab14 and lipid droplets in CNE2-IR and HK1-IR cells and parental cells at 24 h after exposure to 4 Gy IR (red: Ra b14; green: BODIPY 493/503; blue: DAPI; scale bar = 25 μm). (G) Confocal microscopy analysis of the co-localization of CPT1A, Rab14 and mitochondria in the groups indicated in (F) (red: Rab14; green: BODIPY 493/503; blue: DAPI; scale bar = 25 μm).
Figure Legend Snippet: The binding of CPT1A to Rab14 is enhanced in radiation-resistant NPC cells, which might promote the contact of lipid droplet and mitochondria in these cells. (A) Immunoprecipitation analysis showing the binding of CPT1A and Rab14 in CNE2-IR and HK1-IR compared with parental cells. IgG served as a negative control. (B) Immunoprecipitation analysis showing the binding of CPT1A and Rab14 in CNE2 and HK1 cells stably expressing CPT1A-V5 or empty vector. IgG served as a negative control. (C) Proximity ligation assay indicating the interaction of CPT1A and Rab14 in CNE2-IR and HK1-IR compared with parental cells at 24 h after exposure to 4 Gy IR (red: PLA positive signal; blue: DAPI, scale bar = 25 μm). (D) Based on the prediction of Fd-DCA, the N terminal of CPT1A was found to bind with the identified Site 2 of Rab14 ( left ) and the predicted top 20 strongest coupled interacting residue pairs (interacting residue contacts) of these two proteins (orange stick bonds, right ). (E) Immunoblot analysis showing translocation of CPT1A to mitochondria in CNE2 and CNE2-IR cells transfected with a Rab14 siRNA pool or negative siRNA at 24 h after exposure to 4 Gy IR. β-Actin was used as a control to confirm equal loading of protein and Hsp60 served as a control to confirm equal loading of mitochondrial fractions. (F) Confocal microscopy analysis of the co-localization of Rab14 and lipid droplets in CNE2-IR and HK1-IR cells and parental cells at 24 h after exposure to 4 Gy IR (red: Ra b14; green: BODIPY 493/503; blue: DAPI; scale bar = 25 μm). (G) Confocal microscopy analysis of the co-localization of CPT1A, Rab14 and mitochondria in the groups indicated in (F) (red: Rab14; green: BODIPY 493/503; blue: DAPI; scale bar = 25 μm).

Techniques Used: Binding Assay, Immunoprecipitation, Negative Control, Stable Transfection, Expressing, Plasmid Preparation, Proximity Ligation Assay, Translocation Assay, Transfection, Confocal Microscopy

Etomoxir decreases the binding of CPT1A and Rab14, which attenuates fatty acid trafficking in radiation-resistant NPC cells. (A) Immunoprecipitation analysis showing the binding of CPT1A and Rab14 in CNE2-IR and HK1-IR compared with parental cells at 24 h after exposure to 4 Gy IR or Etomoxir (80 μM). IgG served as a negative control. (B) Confocal microscopy analysis of morphologies and subcellular location of free fatty acids in groups indicated in (A) (red: Mito Tracker Red; green: BODIPY C16; blue: DAPI; scale bar = 25 μm). (C) Pearson's coefficient analysis representing relative cellular co-localization of free fatty acids overlapping with mitochondria in groups indicated in (A). Images were analyzed using Image J software. Data are expressed as mean values ± S.D. of 12 independent cells analyzed by microscopy for each group (* p
Figure Legend Snippet: Etomoxir decreases the binding of CPT1A and Rab14, which attenuates fatty acid trafficking in radiation-resistant NPC cells. (A) Immunoprecipitation analysis showing the binding of CPT1A and Rab14 in CNE2-IR and HK1-IR compared with parental cells at 24 h after exposure to 4 Gy IR or Etomoxir (80 μM). IgG served as a negative control. (B) Confocal microscopy analysis of morphologies and subcellular location of free fatty acids in groups indicated in (A) (red: Mito Tracker Red; green: BODIPY C16; blue: DAPI; scale bar = 25 μm). (C) Pearson's coefficient analysis representing relative cellular co-localization of free fatty acids overlapping with mitochondria in groups indicated in (A). Images were analyzed using Image J software. Data are expressed as mean values ± S.D. of 12 independent cells analyzed by microscopy for each group (* p

Techniques Used: Binding Assay, Immunoprecipitation, Negative Control, Confocal Microscopy, Software, Microscopy

A schematic to illustrate CPT1A-mediated radiation resistance in NPC. (A) The enhanced CPT1A expression and the CPT1A-Rab14 interaction promote fatty acid trafficking between lipid droplets and mitochondria, which facilitates fatty acid utilization and maximizes ATP production, leading to resistance to radiation. (B) Blockage of CPT1A attenuates fatty acid trafficking and FAO, causes lipid accumulation and energy stress, which induces mitochondrial apoptosis and re-sensitizes NPC cells to radiation.
Figure Legend Snippet: A schematic to illustrate CPT1A-mediated radiation resistance in NPC. (A) The enhanced CPT1A expression and the CPT1A-Rab14 interaction promote fatty acid trafficking between lipid droplets and mitochondria, which facilitates fatty acid utilization and maximizes ATP production, leading to resistance to radiation. (B) Blockage of CPT1A attenuates fatty acid trafficking and FAO, causes lipid accumulation and energy stress, which induces mitochondrial apoptosis and re-sensitizes NPC cells to radiation.

Techniques Used: Expressing

Knockdown of Rab14 re-sensitizes CPT1A-overexpressing NPC cells to radiation therapy, suppresses fatty acid trafficking from lipid droplets into mitochondria and induces accumulation of lipid droplets in these cells. (A) Colony formation assay showing survival fractions of CNE2-EV, CNE2-CPT1A, HK1-EV and HK1-CPT1A cells transfected with a Rab14 siRNA pool or negative siRNA after exposure to 4 Gy IR. Survival fractions were calculated by comparing the colony number of each treatment group with untreated groups. Results are plotted as the mean survival fraction ± S.D. of 3 independent experiments (* p
Figure Legend Snippet: Knockdown of Rab14 re-sensitizes CPT1A-overexpressing NPC cells to radiation therapy, suppresses fatty acid trafficking from lipid droplets into mitochondria and induces accumulation of lipid droplets in these cells. (A) Colony formation assay showing survival fractions of CNE2-EV, CNE2-CPT1A, HK1-EV and HK1-CPT1A cells transfected with a Rab14 siRNA pool or negative siRNA after exposure to 4 Gy IR. Survival fractions were calculated by comparing the colony number of each treatment group with untreated groups. Results are plotted as the mean survival fraction ± S.D. of 3 independent experiments (* p

Techniques Used: Colony Assay, Transfection

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    Millipore anti rab14 c terminal antibody
    Mutation of serines 580 and 582 does not affect the localization of RCP with endogenous Rab11 or <t>Rab14.</t> HeLa cells expressing the indicated GFP-RCP constructs were fixed and immunolabeled with antibodies that recognize endogenous Rab11 ( A ) and Rab14 (
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    Mutation of serines 580 and 582 does not affect the localization of RCP with endogenous Rab11 or Rab14. HeLa cells expressing the indicated GFP-RCP constructs were fixed and immunolabeled with antibodies that recognize endogenous Rab11 ( A ) and Rab14 (

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: Mutation of serines 580 and 582 does not affect the localization of RCP with endogenous Rab11 or Rab14. HeLa cells expressing the indicated GFP-RCP constructs were fixed and immunolabeled with antibodies that recognize endogenous Rab11 ( A ) and Rab14 (

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques: Mutagenesis, Expressing, Construct, Immunolabeling

    RCP displays greater colocalization with Rab11. A , HeLa cells were fixed and co-labeled with antibodies that recognize endogenous RCP and Rab11 or Rab14. B, quantitative analysis of the colocalization coefficients of endogenous RCP with endogenous Rab11

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: RCP displays greater colocalization with Rab11. A , HeLa cells were fixed and co-labeled with antibodies that recognize endogenous RCP and Rab11 or Rab14. B, quantitative analysis of the colocalization coefficients of endogenous RCP with endogenous Rab11

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques: Labeling

    Rab14 interacts with the RBD of RCP. A, schematic of RCP indicating conserved domains and the location of mutated residues. B, ClustalW alignment of the C-terminal region of the class I FIPs. C, far Western protein-protein interaction assays. Equal amounts

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: Rab14 interacts with the RBD of RCP. A, schematic of RCP indicating conserved domains and the location of mutated residues. B, ClustalW alignment of the C-terminal region of the class I FIPs. C, far Western protein-protein interaction assays. Equal amounts

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques: Western Blot

    Isothermal titration calorimetry of Rab14/effector interactions. A, raw data ( top panel ) and integrated heats ( bottom panel ) of wtRCP(581–649) (600 μ m ) injected into 30 μ m Rab14(2–175). B, injection of 600 μ m Rab14(2–175)

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: Isothermal titration calorimetry of Rab14/effector interactions. A, raw data ( top panel ) and integrated heats ( bottom panel ) of wtRCP(581–649) (600 μ m ) injected into 30 μ m Rab14(2–175). B, injection of 600 μ m Rab14(2–175)

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques: Isothermal Titration Calorimetry, Injection

    Rab11 recruits RCP to membranes. A, HeLa cells were transfected with control siRNA (siFLuc) or siRNA-duplexes targeting Rab11 and Rab14 for 72 h. The cells were transfected with GFP-RCP WT for the final 24 h and fixed. Single 0.4-μm sections were

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: Rab11 recruits RCP to membranes. A, HeLa cells were transfected with control siRNA (siFLuc) or siRNA-duplexes targeting Rab11 and Rab14 for 72 h. The cells were transfected with GFP-RCP WT for the final 24 h and fixed. Single 0.4-μm sections were

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques: Transfection

    Structure of the Rab14-RCP complex. A, ribbon model of Rab14 ( gray ) in complex with RCP homodimer ( yellow and orange ). The switch regions are indicated in red and blue ; GTP is a green stick model, and Mg 2+ is shown as a sphere. B , superposition of Rab14

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: Structure of the Rab14-RCP complex. A, ribbon model of Rab14 ( gray ) in complex with RCP homodimer ( yellow and orange ). The switch regions are indicated in red and blue ; GTP is a green stick model, and Mg 2+ is shown as a sphere. B , superposition of Rab14

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques:

    RCP and Rab14 play a role in neuritogenesis in mouse N2a neuroblastoma cells. A, N2a cells were transfected with the indicated GFP fusion constructs ( green ) for 24 h. Cells were then incubated for a further 24 h in serum-free medium to induce cell differentiation,

    Journal: The Journal of Biological Chemistry

    Article Title: Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP) *

    doi: 10.1074/jbc.M114.612366

    Figure Lengend Snippet: RCP and Rab14 play a role in neuritogenesis in mouse N2a neuroblastoma cells. A, N2a cells were transfected with the indicated GFP fusion constructs ( green ) for 24 h. Cells were then incubated for a further 24 h in serum-free medium to induce cell differentiation,

    Article Snippet: The antibodies used were rabbit anti-Rab14 (R0656), mouse anti-α-tubulin (Thr-5168), and chicken anti-RCP (GW21574A) from Sigma.

    Techniques: Transfection, Construct, Incubation, Cell Differentiation

    Rab14 knock-down increases time required for cells to undergo abscission. (A) Individual images taken from time-lapse analysis of dividing Rab14-KO cell (also see Supplemental Movie 3). (B) Control, Rab14-KD and Rab11a/b-KD cells were fixed and stained with anti-tubulin antibodies to identify cells in telophase. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control, Rab14-KD, Rab11a/b-KD, Rab14/11-DKD and Rab14-KO cells were analysed by time-lapse microscopy to measure the time needed to complete division (starting from metaphase). Where indicated, cells were transfected with GFP-Rab11a or GFP-Rab14. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Control or Rab14-KO cells were incubated in the absence or presence of 4 nM Latrunculin A and multi-nucleated cells counted to assess their ability to complete cytokinesis. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (F-G) Control or Rab14-KO HeLa cells were fixed and stained with phalloidin-596 and anti-tubulin antibodies. The fluorescence of phalloidin-596 at the ICB was then evaluated. The quantification is shown at panel (F). Data shown are the means and standard deviations derived from three independent experiments.

    Journal: bioRxiv

    Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

    doi: 10.1101/2020.04.21.052449

    Figure Lengend Snippet: Rab14 knock-down increases time required for cells to undergo abscission. (A) Individual images taken from time-lapse analysis of dividing Rab14-KO cell (also see Supplemental Movie 3). (B) Control, Rab14-KD and Rab11a/b-KD cells were fixed and stained with anti-tubulin antibodies to identify cells in telophase. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control, Rab14-KD, Rab11a/b-KD, Rab14/11-DKD and Rab14-KO cells were analysed by time-lapse microscopy to measure the time needed to complete division (starting from metaphase). Where indicated, cells were transfected with GFP-Rab11a or GFP-Rab14. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Control or Rab14-KO cells were incubated in the absence or presence of 4 nM Latrunculin A and multi-nucleated cells counted to assess their ability to complete cytokinesis. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (F-G) Control or Rab14-KO HeLa cells were fixed and stained with phalloidin-596 and anti-tubulin antibodies. The fluorescence of phalloidin-596 at the ICB was then evaluated. The quantification is shown at panel (F). Data shown are the means and standard deviations derived from three independent experiments.

    Article Snippet: Antibodies and plasmidsThe following antibodies used for immunofluorescence and western blots: acetylated-α-tubulin (Cell Signalling, D20G3, IF 1:200, WB 1:1000), Rab14 (Sigma-Aldrich, R0656, IF 1:100, WB 1:500), FLAG (Sigma-Aldrich, F1804, IF 1:100, WB 1:1000) and MKLP1 (Thermo Fisher, PA5-31773, IF 1:200).

    Techniques: Staining, Derivative Assay, Time-lapse Microscopy, Transfection, Incubation, Fluorescence

    Schematic representation of the proposed function for Rab14/MACF2 complex. MACF2 is recruited to the central spindle microtubules either by directly binding to microtubule bundles or by co-binding to CAMSAP3, the protein that interacts with microtubule minus-ends. MACF2 then interacts with Rab14, thus, recruiting Rab14-containing early endosomes to the central spindle. Rab11/Fip3 recycling endosomes then buds from early endosomes and is delivered to the abscission site via kinesin-2 molecular motor.

    Journal: bioRxiv

    Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

    doi: 10.1101/2020.04.21.052449

    Figure Lengend Snippet: Schematic representation of the proposed function for Rab14/MACF2 complex. MACF2 is recruited to the central spindle microtubules either by directly binding to microtubule bundles or by co-binding to CAMSAP3, the protein that interacts with microtubule minus-ends. MACF2 then interacts with Rab14, thus, recruiting Rab14-containing early endosomes to the central spindle. Rab11/Fip3 recycling endosomes then buds from early endosomes and is delivered to the abscission site via kinesin-2 molecular motor.

    Article Snippet: Antibodies and plasmidsThe following antibodies used for immunofluorescence and western blots: acetylated-α-tubulin (Cell Signalling, D20G3, IF 1:200, WB 1:1000), Rab14 (Sigma-Aldrich, R0656, IF 1:100, WB 1:500), FLAG (Sigma-Aldrich, F1804, IF 1:100, WB 1:1000) and MKLP1 (Thermo Fisher, PA5-31773, IF 1:200).

    Techniques: Binding Assay

    Rab14 binds to MACF2/CAMSAP3 complex. (A) Putative Rab14-interacting proteins identified in FLAG-Rab14 immunoprecipitation and proteomic analysis. (B) Lysates from HeLa cells expressing endogenously labelled GFP-Rab14 were incubated with either glutathione beads coated with GST or GST-anti-GFP-nanobody. The beads were then washed and bound protein analysed by western blotting. (C-D) HeLa cells transfected with Halo-MACF2 were fixed and stained with Halo ligand and anti-tubulin antibodies. (E-F) Control (E) or Rab14-KO (F) HeLa cells were transfected with GFP-CAMSAP3, then fixed and stained with anti-tubulin antibodies.

    Journal: bioRxiv

    Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

    doi: 10.1101/2020.04.21.052449

    Figure Lengend Snippet: Rab14 binds to MACF2/CAMSAP3 complex. (A) Putative Rab14-interacting proteins identified in FLAG-Rab14 immunoprecipitation and proteomic analysis. (B) Lysates from HeLa cells expressing endogenously labelled GFP-Rab14 were incubated with either glutathione beads coated with GST or GST-anti-GFP-nanobody. The beads were then washed and bound protein analysed by western blotting. (C-D) HeLa cells transfected with Halo-MACF2 were fixed and stained with Halo ligand and anti-tubulin antibodies. (E-F) Control (E) or Rab14-KO (F) HeLa cells were transfected with GFP-CAMSAP3, then fixed and stained with anti-tubulin antibodies.

    Article Snippet: Antibodies and plasmidsThe following antibodies used for immunofluorescence and western blots: acetylated-α-tubulin (Cell Signalling, D20G3, IF 1:200, WB 1:1000), Rab14 (Sigma-Aldrich, R0656, IF 1:100, WB 1:500), FLAG (Sigma-Aldrich, F1804, IF 1:100, WB 1:1000) and MKLP1 (Thermo Fisher, PA5-31773, IF 1:200).

    Techniques: Immunoprecipitation, Expressing, Incubation, Western Blot, Transfection, Staining

    Rab14 is enriched at early endosomes located at the minus-ends of central spindle microtubules. (A-C) HeLa cells expressing GFP-tagged endogenous Rab14 were fixed and stained with anti-tubulin (A), anti-EEA1 (B) or anti-Fip1 (C) antibodies. Asterisks mark the ICB. (D-E) HeLa cells in telophase were fixed and stained with anti-Rab14 (D and E), anti-tubulin (D) or anti-Fip1 (E) antibodies. (E) Schematic representation of proposed endocytic Rab14 localization. (F) HeLa cells in interphase were fixed and stained with anti-Fip1 and anti-Rab14 antibodies.

    Journal: bioRxiv

    Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

    doi: 10.1101/2020.04.21.052449

    Figure Lengend Snippet: Rab14 is enriched at early endosomes located at the minus-ends of central spindle microtubules. (A-C) HeLa cells expressing GFP-tagged endogenous Rab14 were fixed and stained with anti-tubulin (A), anti-EEA1 (B) or anti-Fip1 (C) antibodies. Asterisks mark the ICB. (D-E) HeLa cells in telophase were fixed and stained with anti-Rab14 (D and E), anti-tubulin (D) or anti-Fip1 (E) antibodies. (E) Schematic representation of proposed endocytic Rab14 localization. (F) HeLa cells in interphase were fixed and stained with anti-Fip1 and anti-Rab14 antibodies.

    Article Snippet: Antibodies and plasmidsThe following antibodies used for immunofluorescence and western blots: acetylated-α-tubulin (Cell Signalling, D20G3, IF 1:200, WB 1:1000), Rab14 (Sigma-Aldrich, R0656, IF 1:100, WB 1:500), FLAG (Sigma-Aldrich, F1804, IF 1:100, WB 1:1000) and MKLP1 (Thermo Fisher, PA5-31773, IF 1:200).

    Techniques: Expressing, Staining

    Rab14 is required for targeting Rab11-endosomes to the central spindle microtubules. (A-B) Control or Rab14-KO HeLa cells were fixed and stained with anti-tubulin and anti-Fip1 antibodies. Line-scan analysis of Fip1 fluorescence was then performed in telophase cells (see line in panel A). Asterisks mark the ICB. Data shown in (B) are the means and standard deviations derived from line-scans of four different cells. (C-D) Time-lapse images of dividing control or Rab14-KO cells expressing GFP-Fip3. Asterisks mark the ICB.

    Journal: bioRxiv

    Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

    doi: 10.1101/2020.04.21.052449

    Figure Lengend Snippet: Rab14 is required for targeting Rab11-endosomes to the central spindle microtubules. (A-B) Control or Rab14-KO HeLa cells were fixed and stained with anti-tubulin and anti-Fip1 antibodies. Line-scan analysis of Fip1 fluorescence was then performed in telophase cells (see line in panel A). Asterisks mark the ICB. Data shown in (B) are the means and standard deviations derived from line-scans of four different cells. (C-D) Time-lapse images of dividing control or Rab14-KO cells expressing GFP-Fip3. Asterisks mark the ICB.

    Article Snippet: Antibodies and plasmidsThe following antibodies used for immunofluorescence and western blots: acetylated-α-tubulin (Cell Signalling, D20G3, IF 1:200, WB 1:1000), Rab14 (Sigma-Aldrich, R0656, IF 1:100, WB 1:500), FLAG (Sigma-Aldrich, F1804, IF 1:100, WB 1:1000) and MKLP1 (Thermo Fisher, PA5-31773, IF 1:200).

    Techniques: Staining, Fluorescence, Derivative Assay, Expressing

    Rab14 is required for completion of cytokinesis. (A) Endocytic Rab GTPases present in post-mitotic MB proteome. (B) HeLa cells were transfected with dominant-negative mutants of various MB-associated Rabs. Multi-nucleated cells were then counted and expressed as percentage of total number of cells. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control and Rab14-KD cells were transfected with various GFP-tagged Rabs. The ability of overexpression to decrease multi-nucleation was then analysed. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Individual images taken from time-lapse analysis of dividing control (top row) and Rab14-KO (bottom row) cells (also see Movies 1 2).

    Journal: bioRxiv

    Article Title: Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

    doi: 10.1101/2020.04.21.052449

    Figure Lengend Snippet: Rab14 is required for completion of cytokinesis. (A) Endocytic Rab GTPases present in post-mitotic MB proteome. (B) HeLa cells were transfected with dominant-negative mutants of various MB-associated Rabs. Multi-nucleated cells were then counted and expressed as percentage of total number of cells. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (C-D) Control and Rab14-KD cells were transfected with various GFP-tagged Rabs. The ability of overexpression to decrease multi-nucleation was then analysed. Data shown are the means and standard deviations derived from three independent experiments. n is total number of cells counted. (E) Individual images taken from time-lapse analysis of dividing control (top row) and Rab14-KO (bottom row) cells (also see Movies 1 2).

    Article Snippet: Antibodies and plasmidsThe following antibodies used for immunofluorescence and western blots: acetylated-α-tubulin (Cell Signalling, D20G3, IF 1:200, WB 1:1000), Rab14 (Sigma-Aldrich, R0656, IF 1:100, WB 1:500), FLAG (Sigma-Aldrich, F1804, IF 1:100, WB 1:1000) and MKLP1 (Thermo Fisher, PA5-31773, IF 1:200).

    Techniques: Transfection, Dominant Negative Mutation, Derivative Assay, Over Expression

    Vps13B specifically binds to Rab14 and Rab6 and to PtdIns(3)P. a , b Association of endosomal Rab proteins with Vps13B, revealed by co-immunoprecipitation of endogenous Vps13B with GFP (or mRFP)-tagged Rab proteins. a GFP (or mRFP)-tagged proteins were precipitated from cell lysates using a GFP-trap or anti-mRFP antibody. Bound proteins were analyzed by immunoblotting for Vps13B, GFP, and mRFP. b Cross-linked cells were lysed and immunoprecipitated with anti-Vps13B antibody. The interaction of Rab proteins was detected and indicated Rab protein antibodies. c Coimmunoprecipitation of VPS13B with mRFP-Rab14-WT, GTP-locked mRFP-Rab14Q70L (QL), or GDP-locked mRFP-Rab14S25N (SN) expressed in HeLa cells. Anti-RFP antibody was used for immunoprecipitation of mRFP-tagged Rab14 proteins. There was no significant difference in the binding of Vps13B to the Rab14 variants. d Vps13B co-localizes with Rab6 and Rab14 upon expression in HeLa cells. Top panel: cells were transfected with mRFP-Rab14 and Vps13B-GFP and analyzed for the presence of GFP and mRFP, respectively, by fluorescence microscopy. Bottom panel: cells were transfected with GFP-Rab6. In this case, the distribution of endogenous Vps13B was monitored by immunocytochemistry. White arrows in the upper inserts show vesicles positive for both Vps13B and Rab14. Bottom panels show line scans on the lines in the upper panels and the percentage of colocalization of Rab14 with Vps13B. Scale bar, 10 µm. e PIP strip-binding assay of Vps13B. Perinuclear supernatant from Vps13B-GFP overexpressing cells was incubated with a membrane strip containing the phosphoinositides as indicated and probed for bound Vps13B using an anti-GFP antibody. f Binding of Rhodamine-labeled liposomes to Vps13B-GFP that was immobilized on magnetic Dynabeads. After incubation, the beads were washed, and bound liposomes were detected by Rhodamine fluorescence. Binding was only detectable when Vps13B-GFP was bound to the beads and when the liposomes contained 5% PtdIns(3)P. Error bars indicate S.E.M., *** P

    Journal: Nature Communications

    Article Title: SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors

    doi: 10.1038/s41467-019-09617-9

    Figure Lengend Snippet: Vps13B specifically binds to Rab14 and Rab6 and to PtdIns(3)P. a , b Association of endosomal Rab proteins with Vps13B, revealed by co-immunoprecipitation of endogenous Vps13B with GFP (or mRFP)-tagged Rab proteins. a GFP (or mRFP)-tagged proteins were precipitated from cell lysates using a GFP-trap or anti-mRFP antibody. Bound proteins were analyzed by immunoblotting for Vps13B, GFP, and mRFP. b Cross-linked cells were lysed and immunoprecipitated with anti-Vps13B antibody. The interaction of Rab proteins was detected and indicated Rab protein antibodies. c Coimmunoprecipitation of VPS13B with mRFP-Rab14-WT, GTP-locked mRFP-Rab14Q70L (QL), or GDP-locked mRFP-Rab14S25N (SN) expressed in HeLa cells. Anti-RFP antibody was used for immunoprecipitation of mRFP-tagged Rab14 proteins. There was no significant difference in the binding of Vps13B to the Rab14 variants. d Vps13B co-localizes with Rab6 and Rab14 upon expression in HeLa cells. Top panel: cells were transfected with mRFP-Rab14 and Vps13B-GFP and analyzed for the presence of GFP and mRFP, respectively, by fluorescence microscopy. Bottom panel: cells were transfected with GFP-Rab6. In this case, the distribution of endogenous Vps13B was monitored by immunocytochemistry. White arrows in the upper inserts show vesicles positive for both Vps13B and Rab14. Bottom panels show line scans on the lines in the upper panels and the percentage of colocalization of Rab14 with Vps13B. Scale bar, 10 µm. e PIP strip-binding assay of Vps13B. Perinuclear supernatant from Vps13B-GFP overexpressing cells was incubated with a membrane strip containing the phosphoinositides as indicated and probed for bound Vps13B using an anti-GFP antibody. f Binding of Rhodamine-labeled liposomes to Vps13B-GFP that was immobilized on magnetic Dynabeads. After incubation, the beads were washed, and bound liposomes were detected by Rhodamine fluorescence. Binding was only detectable when Vps13B-GFP was bound to the beads and when the liposomes contained 5% PtdIns(3)P. Error bars indicate S.E.M., *** P

    Article Snippet: Primary antibodies used were obtained from the following companies: anti-APPL1 (3858), anti-LC3B (4599), and anti-Rab7 (9367) from Cell Signaling; anti-EEA1 (612006) and anti-GM130 (560257) from BD Biosciences; anti-M6PR (ab2733), anti-LAMP1 (ab24170), anti-mitofilin (ab110329), anti-mitofusion2 (ab101055), and anti-cathepsin D (ab6313) from Abcam; anti-Tfn receptor (sc-65882) from Santa Cruz Biotechnology; anti-LBPA (Z-PLBPA) from Echelon; anti-RFP (R10367), anti-Golgin97 (A-21270), anti-Vps52 (PA5–24408), and anti-Rab11 (71–5300) from Thermo Fischer Scientific; anti-PDI (700782) from ABfinity; anti-Vps51 (HPA061447), Vps13B (HPA043865) from Atlas antibodies; anti-Rab6 (10187–2-P) and anti-Rab35 (11329–2-AP) from Proteintech; anti-CD63 (H5C6) from Developmental Studies Hybridoma Bank; anti-Rab14 (R0656) from Sigma-Aldrich; anti-GFP (132002), anti-α-tubulin (302211; 1:10,000 dilution for Western blotting), anti-β-actin (251003; 1:10,000 dilution for Western blotting), anti-Rab5 (108011), anti-Stx6 (110062), anti-Stx13 (110132), anti-syntaxin 4 (110042), anti-syntaxin 16 (110162), and anti-VAMP4 (136002) from Synaptic Systems.

    Techniques: Immunoprecipitation, Binding Assay, Expressing, Transfection, Fluorescence, Microscopy, Immunocytochemistry, Stripping Membranes, Incubation, Labeling