Review

pcdna3 ha arf6 actq67l (Addgene inc)

Addgene inc is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Addgene inc pcdna3 ha arf6 actq67l
$Mechanism of the effects of Oxy210 in RAW264.7 cells. A: The effect of <t>Arf6</t> silencing and of Oxy210 (5 μM) on lipid raft abundance. Plasma membranes isolated from cells labeled with [ 3 H] cholesterol were fractionated by density gradient centrifugation as described in “Methods”. Lipid rafts were defined as fractions with highest [ 3 H] cholesterol and flotillin-1 content; P < 0.05 (Mann-Whitney test), Area under the curve values are shown in . B: The effect of Arf6Q67L overexpression and of Oxy210 (5 μM) on lipid raft abundance. Methodology is the same as in A, P < 0.001 (Mann-Whitney test). Area under the curve values are shown in . C: The effect of Arf6 silencing and overexpression and Oxy210 (5 μM) on cholesterol efflux to apoA-I. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to apoA-I (20 μg/ml) over subsequent 2 h incubation is shown. Mean ± SEM; ∗ P < 0.05; versus siRNA scr or pCMV; (ANOVA, n = 4). D: The effect of Arf6 overexpression and Oxy210 (5 μM) on the ABCA1 abundance. The ABCA1 abundance was quantified by densitometry of western blots (shown in B) and was normalized to the abundance of ABCA1 in untransfected cells. Mean ± SEM; ∗∗ P < 0.01; versus pCMV; (ANOVA, n = 3). E: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of IL6. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). F: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of TNFα. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). G: The effect of Oxy210 (5 μM) on the abundance of Arf6. Quantitated by densitometry of western blots shown in C. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). H: The effect of Oxy210 (5 μM) on the ratio of phosphorylated to total Arf6. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). Quantitated by densitometry of western blots shown in D. I: Binding of Arf6 to Oxy210 in protein lipid overlay assay. Indicated amounts of Oxy210, Arf6 (0.2 μg) or vehicle (TBS) were applied (2 μl/dot) on a nitrocellulose membrane (left) or a commercial membrane with preloaded lipids was used (right). Both membranes were incubated for 1 h in the blocking solution and then overnight at 4 ° C with recombinant human His-Arf6 (1 μg/ml), followed by 2 h incubation with anti-His Tag monoclonal antibody (1:1000). J: The effect of Oxy210 (5 μM) on the abundance of PIP1 and PIP2. Lipids extracted from RAW264.7 cell whole cell lysate or isolated lipid rafts were analyzed using liquid chromatography/mass-spectrometry (lipidomics) as described in “Methods”. All concentrations were normalized to the level of PC. Mean ± SEM; ∗ P < 0.05; versus vehicle (n = 4, t -test). LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; apoA-I, apolipoprotein A-I; ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.$
Pcdna3 Ha Arf6 Actq67l, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 ha arf6 actq67l/product/Addgene inc
Average 92 stars, based on 8 article reviews

pcdna3 ha arf6 actq67l - by Bioz Stars, 2026-04

92/100 stars

Images

1) Product Images from "Targeting the ARF6-dependent recycling pathway to alter lipid rafts and reduce inflammation"

Article Title: Targeting the ARF6-dependent recycling pathway to alter lipid rafts and reduce inflammation

Journal: Journal of Lipid Research

doi: 10.1016/j.jlr.2025.100900

$Mechanism of the effects of Oxy210 in RAW264.7 cells. A: The effect of Arf6 silencing and of Oxy210 (5 μM) on lipid raft abundance. Plasma membranes isolated from cells labeled with [ 3 H] cholesterol were fractionated by density gradient centrifugation as described in “Methods”. Lipid rafts were defined as fractions with highest [ 3 H] cholesterol and flotillin-1 content; P < 0.05 (Mann-Whitney test), Area under the curve values are shown in . B: The effect of Arf6Q67L overexpression and of Oxy210 (5 μM) on lipid raft abundance. Methodology is the same as in A, P < 0.001 (Mann-Whitney test). Area under the curve values are shown in . C: The effect of Arf6 silencing and overexpression and Oxy210 (5 μM) on cholesterol efflux to apoA-I. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to apoA-I (20 μg/ml) over subsequent 2 h incubation is shown. Mean ± SEM; ∗ P < 0.05; versus siRNA scr or pCMV; (ANOVA, n = 4). D: The effect of Arf6 overexpression and Oxy210 (5 μM) on the ABCA1 abundance. The ABCA1 abundance was quantified by densitometry of western blots (shown in B) and was normalized to the abundance of ABCA1 in untransfected cells. Mean ± SEM; ∗∗ P < 0.01; versus pCMV; (ANOVA, n = 3). E: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of IL6. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). F: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of TNFα. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). G: The effect of Oxy210 (5 μM) on the abundance of Arf6. Quantitated by densitometry of western blots shown in C. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). H: The effect of Oxy210 (5 μM) on the ratio of phosphorylated to total Arf6. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). Quantitated by densitometry of western blots shown in D. I: Binding of Arf6 to Oxy210 in protein lipid overlay assay. Indicated amounts of Oxy210, Arf6 (0.2 μg) or vehicle (TBS) were applied (2 μl/dot) on a nitrocellulose membrane (left) or a commercial membrane with preloaded lipids was used (right). Both membranes were incubated for 1 h in the blocking solution and then overnight at 4 ° C with recombinant human His-Arf6 (1 μg/ml), followed by 2 h incubation with anti-His Tag monoclonal antibody (1:1000). J: The effect of Oxy210 (5 μM) on the abundance of PIP1 and PIP2. Lipids extracted from RAW264.7 cell whole cell lysate or isolated lipid rafts were analyzed using liquid chromatography/mass-spectrometry (lipidomics) as described in “Methods”. All concentrations were normalized to the level of PC. Mean ± SEM; ∗ P < 0.05; versus vehicle (n = 4, t -test). LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; apoA-I, apolipoprotein A-I; ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.$
Figure Legend Snippet: Mechanism of the effects of Oxy210 in RAW264.7 cells. A: The effect of Arf6 silencing and of Oxy210 (5 μM) on lipid raft abundance. Plasma membranes isolated from cells labeled with [ 3 H] cholesterol were fractionated by density gradient centrifugation as described in “Methods”. Lipid rafts were defined as fractions with highest [ 3 H] cholesterol and flotillin-1 content; P < 0.05 (Mann-Whitney test), Area under the curve values are shown in . B: The effect of Arf6Q67L overexpression and of Oxy210 (5 μM) on lipid raft abundance. Methodology is the same as in A, P < 0.001 (Mann-Whitney test). Area under the curve values are shown in . C: The effect of Arf6 silencing and overexpression and Oxy210 (5 μM) on cholesterol efflux to apoA-I. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to apoA-I (20 μg/ml) over subsequent 2 h incubation is shown. Mean ± SEM; ∗ P < 0.05; versus siRNA scr or pCMV; (ANOVA, n = 4). D: The effect of Arf6 overexpression and Oxy210 (5 μM) on the ABCA1 abundance. The ABCA1 abundance was quantified by densitometry of western blots (shown in B) and was normalized to the abundance of ABCA1 in untransfected cells. Mean ± SEM; ∗∗ P < 0.01; versus pCMV; (ANOVA, n = 3). E: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of IL6. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). F: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of TNFα. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). G: The effect of Oxy210 (5 μM) on the abundance of Arf6. Quantitated by densitometry of western blots shown in C. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). H: The effect of Oxy210 (5 μM) on the ratio of phosphorylated to total Arf6. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). Quantitated by densitometry of western blots shown in D. I: Binding of Arf6 to Oxy210 in protein lipid overlay assay. Indicated amounts of Oxy210, Arf6 (0.2 μg) or vehicle (TBS) were applied (2 μl/dot) on a nitrocellulose membrane (left) or a commercial membrane with preloaded lipids was used (right). Both membranes were incubated for 1 h in the blocking solution and then overnight at 4 ° C with recombinant human His-Arf6 (1 μg/ml), followed by 2 h incubation with anti-His Tag monoclonal antibody (1:1000). J: The effect of Oxy210 (5 μM) on the abundance of PIP1 and PIP2. Lipids extracted from RAW264.7 cell whole cell lysate or isolated lipid rafts were analyzed using liquid chromatography/mass-spectrometry (lipidomics) as described in “Methods”. All concentrations were normalized to the level of PC. Mean ± SEM; ∗ P < 0.05; versus vehicle (n = 4, t -test). LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; apoA-I, apolipoprotein A-I; ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.

Techniques Used: Clinical Proteomics, Isolation, Labeling, Gradient Centrifugation, MANN-WHITNEY, Over Expression, Incubation, Western Blot, Inhibition, Binding Assay, Protein-lipid Overlay Assay (PLOA), Membrane, Blocking Assay, Recombinant, Liquid Chromatography, Mass Spectrometry

Schematic representation of the proposed mechanism of action of Oxy210. Plasma membrane components are constantly recycling between the membrane and intracellular compartments. During recycling they pass through a checkpoint in late endosomes, where components of “disordered” regions of plasma membrane are directed to lysosomes, while “ordered” ensembles are directed to the plasma membrane lipid rafts. Small GTPases, including Arf6, are proposed to play key role in this regulatory checkpoint through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which catalyzes conversion of PIP1 to PIP2. Arf6 is also involved in sorting of internalized ABCA1; however, it works in an opposite direction trafficking ABCA1 for degradation in lysosomes. We propose that Oxy210 reduces the abundance and activity of Arf6, which in turn decreases the activity of PIP5K, the abundance of PIP2 and the recycling of lipid rafts to the plasma membrane. Suppression of Arf6 by Oxy210 also inhibited internalization and degradation of ABCA1, increasing its abundance on the cell surface and consequently increasing the rate of cholesterol efflux. ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.

Figure Legend Snippet: Schematic representation of the proposed mechanism of action of Oxy210. Plasma membrane components are constantly recycling between the membrane and intracellular compartments. During recycling they pass through a checkpoint in late endosomes, where components of “disordered” regions of plasma membrane are directed to lysosomes, while “ordered” ensembles are directed to the plasma membrane lipid rafts. Small GTPases, including Arf6, are proposed to play key role in this regulatory checkpoint through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which catalyzes conversion of PIP1 to PIP2. Arf6 is also involved in sorting of internalized ABCA1; however, it works in an opposite direction trafficking ABCA1 for degradation in lysosomes. We propose that Oxy210 reduces the abundance and activity of Arf6, which in turn decreases the activity of PIP5K, the abundance of PIP2 and the recycling of lipid rafts to the plasma membrane. Suppression of Arf6 by Oxy210 also inhibited internalization and degradation of ABCA1, increasing its abundance on the cell surface and consequently increasing the rate of cholesterol efflux. ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.

Techniques Used: Clinical Proteomics, Membrane, Activation Assay, Activity Assay, Binding Assay

Similar Products

Image Search Results

Journal: Journal of Lipid Research

Article Title: Targeting the ARF6-dependent recycling pathway to alter lipid rafts and reduce inflammation

doi: 10.1016/j.jlr.2025.100900

Figure Lengend Snippet: Mechanism of the effects of Oxy210 in RAW264.7 cells. A: The effect of Arf6 silencing and of Oxy210 (5 μM) on lipid raft abundance. Plasma membranes isolated from cells labeled with [ 3 H] cholesterol were fractionated by density gradient centrifugation as described in “Methods”. Lipid rafts were defined as fractions with highest [ 3 H] cholesterol and flotillin-1 content; P < 0.05 (Mann-Whitney test), Area under the curve values are shown in . B: The effect of Arf6Q67L overexpression and of Oxy210 (5 μM) on lipid raft abundance. Methodology is the same as in A, P < 0.001 (Mann-Whitney test). Area under the curve values are shown in . C: The effect of Arf6 silencing and overexpression and Oxy210 (5 μM) on cholesterol efflux to apoA-I. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to apoA-I (20 μg/ml) over subsequent 2 h incubation is shown. Mean ± SEM; ∗ P < 0.05; versus siRNA scr or pCMV; (ANOVA, n = 4). D: The effect of Arf6 overexpression and Oxy210 (5 μM) on the ABCA1 abundance. The ABCA1 abundance was quantified by densitometry of western blots (shown in B) and was normalized to the abundance of ABCA1 in untransfected cells. Mean ± SEM; ∗∗ P < 0.01; versus pCMV; (ANOVA, n = 3). E: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of IL6. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). F: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of TNFα. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). G: The effect of Oxy210 (5 μM) on the abundance of Arf6. Quantitated by densitometry of western blots shown in C. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). H: The effect of Oxy210 (5 μM) on the ratio of phosphorylated to total Arf6. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). Quantitated by densitometry of western blots shown in D. I: Binding of Arf6 to Oxy210 in protein lipid overlay assay. Indicated amounts of Oxy210, Arf6 (0.2 μg) or vehicle (TBS) were applied (2 μl/dot) on a nitrocellulose membrane (left) or a commercial membrane with preloaded lipids was used (right). Both membranes were incubated for 1 h in the blocking solution and then overnight at 4 ° C with recombinant human His-Arf6 (1 μg/ml), followed by 2 h incubation with anti-His Tag monoclonal antibody (1:1000). J: The effect of Oxy210 (5 μM) on the abundance of PIP1 and PIP2. Lipids extracted from RAW264.7 cell whole cell lysate or isolated lipid rafts were analyzed using liquid chromatography/mass-spectrometry (lipidomics) as described in “Methods”. All concentrations were normalized to the level of PC. Mean ± SEM; ∗ P < 0.05; versus vehicle (n = 4, t -test). LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; apoA-I, apolipoprotein A-I; ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.

Article Snippet: To transfect RAW264.7 cells with control pCMV, pcDNA3 HA Arf6 ActQ67L, or pcDNA3 HA Arf6 DN T27N (Addgene) Lipofectamine LTX with Plus reagent (Invitrogen, #15338100) was used according to manufacturer protocol.

Techniques: Clinical Proteomics, Isolation, Labeling, Gradient Centrifugation, MANN-WHITNEY, Over Expression, Incubation, Western Blot, Inhibition, Binding Assay, Protein-lipid Overlay Assay (PLOA), Membrane, Blocking Assay, Recombinant, Liquid Chromatography, Mass Spectrometry

Journal: Journal of Lipid Research

Article Title: Targeting the ARF6-dependent recycling pathway to alter lipid rafts and reduce inflammation

doi: 10.1016/j.jlr.2025.100900

Figure Lengend Snippet: Schematic representation of the proposed mechanism of action of Oxy210. Plasma membrane components are constantly recycling between the membrane and intracellular compartments. During recycling they pass through a checkpoint in late endosomes, where components of “disordered” regions of plasma membrane are directed to lysosomes, while “ordered” ensembles are directed to the plasma membrane lipid rafts. Small GTPases, including Arf6, are proposed to play key role in this regulatory checkpoint through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which catalyzes conversion of PIP1 to PIP2. Arf6 is also involved in sorting of internalized ABCA1; however, it works in an opposite direction trafficking ABCA1 for degradation in lysosomes. We propose that Oxy210 reduces the abundance and activity of Arf6, which in turn decreases the activity of PIP5K, the abundance of PIP2 and the recycling of lipid rafts to the plasma membrane. Suppression of Arf6 by Oxy210 also inhibited internalization and degradation of ABCA1, increasing its abundance on the cell surface and consequently increasing the rate of cholesterol efflux. ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Activity Assay, Binding Assay

A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).

Article Snippet: Wild‐type, constitutively active, and dominant negative human Arf6, Arf6 Q67L , and Arf6 T27N were amplified from pcDNA3‐HA‐Arf6 (Addgene #10834), pcDNA3‐HA‐Arf6 Q67L (Addgene #10835), or pcDNA3‐HA‐Arf6 T27N (Addgene #10831) using primers WSU0433 and WSU0437 and cloned into either pEGFP‐N1 or pmCherry‐N1 (Clontech) using Eco RI and Kpn I restriction sites to generate pmEGFP‐N1‐Arf6, pEGFP‐N1‐Arf6 Q67L , pEGFP‐N1‐Arf6 T27N or pmCherry‐N1‐Arf6, pmCherry‐N1‐Arf6 Q67L , and pmCherry‐N1‐Arf6 T27N .

Techniques: Transfection, Incubation, Binding Assay, Staining, Marker

Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

Techniques: Transfection, Western Blot, Immunoprecipitation, Magnetic Beads, SDS Page, Control, Activity Assay, Infection, Bacteria

Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

Techniques: Fluorescence, Transfection, Labeling

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet:

Techniques: Derivative Assay, Recombinant, Transduction, Sequencing, Software, cDNA Library Assay, Transformation Assay, Plasmid Preparation, Isolation, Activation Assay

ARL4C, Cytohesin-1, and ARF6 accumulate in the RBX2 mutant, but not in SOCS7−/− hippocampi. (A) TMT-MS of Rbx2cKO–Nes (n = 3) and Rbx2 fl/fl (n = 4) telencephalons (P0) plotted as a ratio of Rbx2cKO-Nes/control. The x axis represents the logarithmic fold ration of Rbx2cKO–Nes/control per protein identified. The y axis represents the logarithmic P value of the t test per protein identified. Proteins whose differentially expression in comparison to control (Rbx2 fl/fl) have a P < 0.01 and accumulated above 1.5 folds (red dots) or reduced lower than 0.666 folds (blue dots) in comparison to control were initially considered for further studies. As expected, RBX2 mutant samples showed lower levels of RBX2 and CUL5 and higher levels of DAB1 in comparison to control. (B) RNA-seq analysis comparing Rbx2cKO–Nes (Mutant; n = 3) and Rbx2 fl/fl (Control; n = 3) telencephalon at P1 was performed to determine transcriptionally differences between genotypes. Ratio of counts per million between Rbx2cKO–Nes and control per gene is plotted. The x axis represents the logarithmic fold ration of Rbx2cKO–Nes/control per gene identified. The y axis represents the logarithmic false discovery rate value (FDR; or q value) calculated by using the Benjamini, Krieger, and Yekutieli method. Only genes with an average count per million (cpm) in control samples of >1 were used for statistical purposes and plotted. No significant transcriptomic differences were observed between genotypes. (C) Western blot analyses validating TMT-MS data in P1 Rbx2cKO–Emx1 and control hippocampi. Arrow indicates pY-DAB1 band in anti–p-Tyr blot. Numbers indicate ratio of proteins in Rbx2 fl/fl (n = 5) vs. Rbx2cKO–Emx1 (n = 3). Statistics, Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Increased binding between CYTH1 and ARF6 in the Rbx2cKO-Emx1 hippocampus in comparison to control. An anti-CYTH1 antibody was used to immunoprecipitated CYTH1 and Western blotting was used to determine the levels of ARF6 coimmunoprecipitated. Representative blot of Rbx2 fl/fl (Ctrl; n = 4) and Rbx2cKO–Emx1 (cKO; n = 3) coimmunoprecipitation is shown. Arrow indicates ARF6 band, * indicates IgG. (E) Elevated ARF6-GTP levels were detected in Rbx2cKO–Emx1 hippocampal lysates by using a GGA3–GST pull-down assay in comparison to control. Numbers indicate ratio of ARF6–GTP detected in Rbx2 fl/fl (n = 3) vs. Rbx2cKO–Emx1 (n = 3). Statistics, Student’s t test. *P < 0.05. (F) ARL4C, CYTH1, and ARF6 did not accumulate in P4 SOCS7 −/− (n= 3) in comparison to controls (SOCS7 +/−; n = 4) hippocampus lysates when analyzed by Western blotting. Statistics, Student’s t test. WCL, whole-cell lysate.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis

doi: 10.1073/pnas.2002749117

Figure Lengend Snippet: ARL4C, Cytohesin-1, and ARF6 accumulate in the RBX2 mutant, but not in SOCS7−/− hippocampi. (A) TMT-MS of Rbx2cKO–Nes (n = 3) and Rbx2 fl/fl (n = 4) telencephalons (P0) plotted as a ratio of Rbx2cKO-Nes/control. The x axis represents the logarithmic fold ration of Rbx2cKO–Nes/control per protein identified. The y axis represents the logarithmic P value of the t test per protein identified. Proteins whose differentially expression in comparison to control (Rbx2 fl/fl) have a P < 0.01 and accumulated above 1.5 folds (red dots) or reduced lower than 0.666 folds (blue dots) in comparison to control were initially considered for further studies. As expected, RBX2 mutant samples showed lower levels of RBX2 and CUL5 and higher levels of DAB1 in comparison to control. (B) RNA-seq analysis comparing Rbx2cKO–Nes (Mutant; n = 3) and Rbx2 fl/fl (Control; n = 3) telencephalon at P1 was performed to determine transcriptionally differences between genotypes. Ratio of counts per million between Rbx2cKO–Nes and control per gene is plotted. The x axis represents the logarithmic fold ration of Rbx2cKO–Nes/control per gene identified. The y axis represents the logarithmic false discovery rate value (FDR; or q value) calculated by using the Benjamini, Krieger, and Yekutieli method. Only genes with an average count per million (cpm) in control samples of >1 were used for statistical purposes and plotted. No significant transcriptomic differences were observed between genotypes. (C) Western blot analyses validating TMT-MS data in P1 Rbx2cKO–Emx1 and control hippocampi. Arrow indicates pY-DAB1 band in anti–p-Tyr blot. Numbers indicate ratio of proteins in Rbx2 fl/fl (n = 5) vs. Rbx2cKO–Emx1 (n = 3). Statistics, Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Increased binding between CYTH1 and ARF6 in the Rbx2cKO-Emx1 hippocampus in comparison to control. An anti-CYTH1 antibody was used to immunoprecipitated CYTH1 and Western blotting was used to determine the levels of ARF6 coimmunoprecipitated. Representative blot of Rbx2 fl/fl (Ctrl; n = 4) and Rbx2cKO–Emx1 (cKO; n = 3) coimmunoprecipitation is shown. Arrow indicates ARF6 band, * indicates IgG. (E) Elevated ARF6-GTP levels were detected in Rbx2cKO–Emx1 hippocampal lysates by using a GGA3–GST pull-down assay in comparison to control. Numbers indicate ratio of ARF6–GTP detected in Rbx2 fl/fl (n = 3) vs. Rbx2cKO–Emx1 (n = 3). Statistics, Student’s t test. *P < 0.05. (F) ARL4C, CYTH1, and ARF6 did not accumulate in P4 SOCS7 −/− (n= 3) in comparison to controls (SOCS7 +/−; n = 4) hippocampus lysates when analyzed by Western blotting. Statistics, Student’s t test. WCL, whole-cell lysate.

Article Snippet: The pSG5–hARL4C construct was a generous gift from Fang-Jen Lee, National Taiwan University, Taiwan ( 4 ). pcDNA3–HA–ARF6 and pcDNA3–HA–ARF6 Q67L were a gift from Thomas Roberts, Harvard Medical School, Boston, MA [Addgene plasmid nos.

Techniques: Mutagenesis, Expressing, RNA Sequencing Assay, Western Blot, Binding Assay, Immunoprecipitation, Pull Down Assay

ARL4C and ARF6 have opposite effects on CRL5-dependent PN overmigration, but the same effects on PN dendrite extension into the slm. (A–E) Representative images of hippocampus electroporated at E15 and collected at P10 showing control (GFP) (A), CUL5 KD (shCUL5) (B), double CUL5/ARL4C KD (shCUL5/shARL4C) (C), double CUL5/CYTH1 KD (shCUL5/shCYTH1) (D), and double CUL5/ARF6 KD (shCUL5/shARF6) (E) transfected PNs. Whereas ARL4C KD rescue the PN overmigration phenotype caused by CUL5 KD, CYTH1 and ARF6 KD exacerbated it. CUL5/ARL4C-, CUL5/ARF6-, and CUL5/CYTH1-KD PNs fail to extend their apical dendrites into the slm in comparison to control or CUL5-KD PNs. The striped lines represent the hippocampal fissure. (F) Quantification of the percentage number of GFP + cells in each strata (Left) and percentage of GFP + cells with normal, disrupted, or multiple apical dendrites (Right). Mean ± SD. Statistics, two-way ANOVA with Tukey’s multiple-comparison test. (G) Overexpression of ARL4C, CYTH1, and DAB1–EGFP by IUE is sufficient to mimic CUL5 KD phenotypes (B). Pink arrowheads indicate PNs with correct position. Yellow solid arrowheads indicate PNs with abnormal apical dendrites. Yellow hollow arrowheads indicate overmigrated PNs. The striped lines represent the hippocampal fissure. (H) Quantification of the percentage number of GFP+ cells in each strata (Upper) and percentage of GFP + cells with normal, disrupted, or multiple apical dendrites (Lower). Mean ± SD. Statistics, two-way ANOVA with Tukey’s multiple comparison test. (I and J) Single ARL4C (shARL4C) (I) or ARF6 (shARF6) (J) KD constructs were cotransfected with GFP by IUE in E15 hippocampi and brains were collected at P10. In both conditions, PNs migrated normally and had a single radially oriented apical dendrite, but failed to extend their dendrites into the slm. The striped lines represent the hippocampal fissure.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis

doi: 10.1073/pnas.2002749117

Figure Lengend Snippet: ARL4C and ARF6 have opposite effects on CRL5-dependent PN overmigration, but the same effects on PN dendrite extension into the slm. (A–E) Representative images of hippocampus electroporated at E15 and collected at P10 showing control (GFP) (A), CUL5 KD (shCUL5) (B), double CUL5/ARL4C KD (shCUL5/shARL4C) (C), double CUL5/CYTH1 KD (shCUL5/shCYTH1) (D), and double CUL5/ARF6 KD (shCUL5/shARF6) (E) transfected PNs. Whereas ARL4C KD rescue the PN overmigration phenotype caused by CUL5 KD, CYTH1 and ARF6 KD exacerbated it. CUL5/ARL4C-, CUL5/ARF6-, and CUL5/CYTH1-KD PNs fail to extend their apical dendrites into the slm in comparison to control or CUL5-KD PNs. The striped lines represent the hippocampal fissure. (F) Quantification of the percentage number of GFP + cells in each strata (Left) and percentage of GFP + cells with normal, disrupted, or multiple apical dendrites (Right). Mean ± SD. Statistics, two-way ANOVA with Tukey’s multiple-comparison test. (G) Overexpression of ARL4C, CYTH1, and DAB1–EGFP by IUE is sufficient to mimic CUL5 KD phenotypes (B). Pink arrowheads indicate PNs with correct position. Yellow solid arrowheads indicate PNs with abnormal apical dendrites. Yellow hollow arrowheads indicate overmigrated PNs. The striped lines represent the hippocampal fissure. (H) Quantification of the percentage number of GFP+ cells in each strata (Upper) and percentage of GFP + cells with normal, disrupted, or multiple apical dendrites (Lower). Mean ± SD. Statistics, two-way ANOVA with Tukey’s multiple comparison test. (I and J) Single ARL4C (shARL4C) (I) or ARF6 (shARF6) (J) KD constructs were cotransfected with GFP by IUE in E15 hippocampi and brains were collected at P10. In both conditions, PNs migrated normally and had a single radially oriented apical dendrite, but failed to extend their dendrites into the slm. The striped lines represent the hippocampal fissure.

Techniques: Transfection, Over Expression, Construct