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Addgene inc pcdna3 ha arf6 q67l

A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, <t>Arf6</t> T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 <t>Q67L</t> ‐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).

Pcdna3 Ha Arf6 Q67l, 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/product/pcdna3+ha+arf6+q67l/pcDNA3+HA+Arf6+ActQ67L+(Plasmid+%2310835)/pmc08488576-290-20-22
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pcdna3 ha arf6 q67l - by Bioz Stars, 2026-10

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1) Product Images from "A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication"

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

Journal: The EMBO Journal

doi: 10.15252/embj.2021107664

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).

Figure Legend 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).

Techniques Used: 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.

Figure Legend 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 Used: 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.

Figure Legend 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 Used: Fluorescence, Transfection, Labeling

Figure Legend Snippet:

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

Dominant Negative Mutation:

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication
Article Snippet: Dominant negative human Rab8a T22N was digested from pEGFP‐C1‐Rab8a T22N (Hattula & Peränen, ) using Eco RI and Bam HI restriction sites and ligated into pmCherry‐C1 (Clontech) to generate pmCherry‐Rab8a T22N . .. 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 . ..

Amplification:

Clone Assay:

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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

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

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

(A) Quantification of invaded cells through Matrigel-coated transwell. ADAP1-WT-expressing cells showed increased invasive capability compared with control and ADAP1-R49K-expressing cells. n = 3–4. *** P < 0.001. P value by unpaired t test is indicated. (B) RT-qPCR analysis of Arf6 mRNA in cells transduced with lentiviral Arf6 or scramble shRNAs. n = 3. (C) Quantification of cell invasion. The ADAP1-induced invasion was reduced by Arf6 knockdown. n = 4. *** P < 0.001. P value by unpaired t test is indicated. (D) Western blot analysis of cell lysates overexpressing ARF6-WT, ARF6-CA, and ADAP1. α-tubulin, loading control. (E) Quantification of invaded cells. ADAP1-induced cell invasion was reduced by ARF6-CA expression. n = 4. * P < 0.05. P value by unpaired t test is indicated. (F) RT-qPCR analysis of human ADAP1 ( hADAP1 ) RNA in SCC-61 cells transduced with lentiviral hADAP1 or scramble shRNAs. n = 3. (G) Western blot analysis of SCC-61 cell lysates transduced with scramble or hADAP1 shRNAs. (H) Quantification of invaded cells. Invasive activity of SCC-61 cells was reduced by hADAP1 knockdown. n = 3. Data are mean ± SD. *** P < 0.001, * P < 0.05. P value by unpaired t test is indicated. ns, not significant.

Journal: Life Science Alliance

Article Title: ADAP1 promotes invasive squamous cell carcinoma progression and predicts patient survival

doi: 10.26508/lsa.201900582

Figure Lengend Snippet: (A) Quantification of invaded cells through Matrigel-coated transwell. ADAP1-WT-expressing cells showed increased invasive capability compared with control and ADAP1-R49K-expressing cells. n = 3–4. *** P < 0.001. P value by unpaired t test is indicated. (B) RT-qPCR analysis of Arf6 mRNA in cells transduced with lentiviral Arf6 or scramble shRNAs. n = 3. (C) Quantification of cell invasion. The ADAP1-induced invasion was reduced by Arf6 knockdown. n = 4. *** P < 0.001. P value by unpaired t test is indicated. (D) Western blot analysis of cell lysates overexpressing ARF6-WT, ARF6-CA, and ADAP1. α-tubulin, loading control. (E) Quantification of invaded cells. ADAP1-induced cell invasion was reduced by ARF6-CA expression. n = 4. * P < 0.05. P value by unpaired t test is indicated. (F) RT-qPCR analysis of human ADAP1 ( hADAP1 ) RNA in SCC-61 cells transduced with lentiviral hADAP1 or scramble shRNAs. n = 3. (G) Western blot analysis of SCC-61 cell lysates transduced with scramble or hADAP1 shRNAs. (H) Quantification of invaded cells. Invasive activity of SCC-61 cells was reduced by hADAP1 knockdown. n = 3. Data are mean ± SD. *** P < 0.001, * P < 0.05. P value by unpaired t test is indicated. ns, not significant.

Article Snippet: For generating ARF6-expression lentiviral vectors, we obtained pcDNA3-Arf6 (WT) and Arf6 (Q67L) from Addgene (plasmid ID 79424 and 79425, respectively) and subcloned PCR-amplified Arf6 fragments into pLKO vector, which was followed by sequence confirmation.

Techniques: Expressing, Control, Quantitative RT-PCR, Transduction, Knockdown, Western Blot, Activity Assay

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pcdna3 ha arf6 q67l (Addgene inc)

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