Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Arf1 and Arf6 promote ventral actin structures formed by acute activation of protein kinase C and Src.

doi: 10.1002/cm.21181

Figure Lengend Snippet: Fig. 1. PMA treated Beas-2b cells form ventral actin waves. (A) Untransfected Beas-2b cells were treated with vehicle (no PMA) or 200 nM PMA for 30 min prior to fixation and staining with rhodamine phalloidin. (B) Beas-2b cells were transfected with plasmids encoding untagged Arf6 Q67L (top) or Arf1 Q71L- GFP (bottom) and treated with 200 nM PMA for 30 min prior to fixation and labeling with antibodies to Arf6 for Arf6 detec- tion and actin. Bars, 10 mm. (C) The fraction of transfected cells with one or more ventral waves was quantified and is expressed as the average percentage obtained from three inde- pendent experiments. Error bars represent standard deviation from the means. GFP was used as a control for transfection. One-way ANOVA test of the PMA-treated control vs. Arf6Q67L- and Arf1Q71L-transfected cells were significant (P < 0.05). (D) Beas-2b cells transfected with Mem-GFP and RFP-LifeAct were imaged as described in Materials and Meth- ods section. PMA was added after 2 min, designated time 0, and frames were captured every 30 sec thereafter. Selected stills from the movie at 22, 6, 12, and 18 min are shown (see Sup- porting Information Movie 1). No membrane folds are associ- ated with actin structures. Bar, 10 mM. Boxed regions are shown at higher magnification below each time point. Bar, 5mM. (E) Beas-2b cells were transfected with plasmids encoding untagged Arf6 Q67L (in background), Mem-GFP to mark vacuolar membranes in transfected cells, and LifeAct-RFP (to visualize actin). A cell was imaged for 20 min; at 5 min, 200 nM PMA was added (arrow) to induce ventral wave forma- tion (Supporting Information Movie 2). (F) Beas-2b cells were transfected with plasmids encoding Arf1-Q71L-RFP and GFP- Actin. A cell was imaged for 15 min; at 5 min 200 nM PMA was added (arrow) to induce ventral wave formation (Support- ing Information Movie 3). Shown for each movie is an image of the entire cell taken at the end of the movie with a square around the region shown in the movie. A series of frames from the movie with time indicated in seconds from the beginning of the movie is also shown. Black arrows indicate when PMA was added. Bars, 10 mm.

Article Snippet: Western blots were carried out using a rabbit polyclonal antibody to Arf1, a rabbit polyclonal antibody to Arf6 (see details above) or a mouse monoclonal antibody to Arf6 (Santa Cruz Biotechnology), and a rabbit antibody to actin (Sigma), and a mouse monoclonal antibody to a-tubulin (DM1A) (Sigma).

Techniques: Staining, Transfection, Labeling, Standard Deviation, Control, Membrane

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Arf1 and Arf6 promote ventral actin structures formed by acute activation of protein kinase C and Src.

doi: 10.1002/cm.21181

Figure Lengend Snippet: Fig. 2. Ventral actin wave formation in Beas-2b cells requires PKC, Src, Rac activity, PIP2, and Arf. (A) Beas-2b cells were treated for 30 min with 200 nM PMA in the absence or pres- ence of 10 mM GF 109303x (inhibitor of PKC), 10 mM PP3 (inactive analog of PP2) or 10 mM PP2 (inhibitor of Src) as indicated. Following PMA treatment, cells were fixed and stained with rhodamine phalloidin as described. Bars,10 mm. (B) Fraction of control or drug-treated cells with one or more ventral waves visible in phalloidin stain was quantified and is expressed as the average percentage from three independent experiments. Error bars represent standard deviation from the mean. Tukey multiple comparison test showed that GF and PP2 differed from the control (P < 0.001). (C) Beas-2b cells express- ing untagged Rac T31N, the p72 PI(4,5)P2 5-phosphatase (myc tagged), Arf6 T27N, or Arf1 T31N-HA were treated with 200 nM PMA 30 min prior to fixation and immunofluores- cence staining as described in the Material and Methods section with an appropriate primary antibody to detect the transfected protein and Alexa 488 conjugated secondary antibody. Cells were costained with rhodamine phalloidin (right panels). (D) Fraction of transfected cells with one or more ventral waves visi- ble in phalloidin stain was quantified and is expressed as the average percentage from three independent experiments. Error bars represent standard deviation from the mean. One way ANOVA test showed all treatments differed from control (P < 0.001). (E) Untransfected Beas-2b cells were left untreated (control) or pretreated for 2 h with 5 mg/ml brefeldin A (BFA). Both sets of cells were treated with 200 nM PMA for 30 min prior to fixation and staining with rhodamine phalloidin and antibody to GM130. The percentage of cells with one or more ventral waves was quantified and is expressed as the average per- centage of three independent experiments. Error bars represent one standard deviation from the mean. Student’s T-test showed control vs. BFA treated was not statistically significant. Bars, 10 mm.

Article Snippet: Western blots were carried out using a rabbit polyclonal antibody to Arf1, a rabbit polyclonal antibody to Arf6 (see details above) or a mouse monoclonal antibody to Arf6 (Santa Cruz Biotechnology), and a rabbit antibody to actin (Sigma), and a mouse monoclonal antibody to a-tubulin (DM1A) (Sigma).

Techniques: Activity Assay, Staining, Control, Standard Deviation, Comparison, Transfection

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Arf1 and Arf6 promote ventral actin structures formed by acute activation of protein kinase C and Src.

doi: 10.1002/cm.21181

Figure Lengend Snippet: Fig. 3. Arf GEFs enhance ventral actin structure formation in Beas-2b cells. (A) Beas 2B cells were transfected with plasmids encoding Flag-EFA6 (Arf6 GEF), or the Arf1 GEFS Flag- ARNO 2G (PIP3 binding), or Flag-ARNO 3G (PIP2 binding). Cells were treated with PMA, fixed and immunostained with antibody against Flag and rhodamine phalloidin. Bar, 10 mm. (B) Percentage of cells exhibiting one or more ventral actin structures was quantified. With the exception of Flag-EFA6, in total, 200 cells were counted from three independent experi- ments. For Flag-EFA6, in total, 123 cells were counted from three independent experiments. Error bars represent standard error for proportional data, P < 0.0001 as determined by Fish- er’s Exact test (two-sided) indicating that GEF expression resulted in increase in cells with ventral actin structures.

Article Snippet: Western blots were carried out using a rabbit polyclonal antibody to Arf1, a rabbit polyclonal antibody to Arf6 (see details above) or a mouse monoclonal antibody to Arf6 (Santa Cruz Biotechnology), and a rabbit antibody to actin (Sigma), and a mouse monoclonal antibody to a-tubulin (DM1A) (Sigma).

Techniques: Transfection, Binding Assay, Expressing

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Arf1 and Arf6 promote ventral actin structures formed by acute activation of protein kinase C and Src.

doi: 10.1002/cm.21181

Figure Lengend Snippet: Fig. 4. Active Arf6 or Arf1 promotes ventral actin structure formation in HeLa cells upon PMA treatment. HeLa cells were trans- fected with plasmids encoding Mem-GFP, FLAG-EFA6, FLAG-EFA6-EK, untagged Arf6 Q67L or Arf1Q71L-GFP. 18 h following transfection, cells were untreated (A) or treated for 30 min with 200 nM PMA (B) prior to fixation and immunofluorescence stain- ing as described in Materials and Methods section. F-actin was labeled with rhodamine phalloidin, and antibodies to FLAG, Arf6 and Arf1 were detected with an Alexa 488-conjugated secondary antibody. In (B) arrow points to ventral ruffles, which are enlarged in inset. Bars, 10 mm. (C) Fraction of cells expressing Mem-GFP, FLAG-EFA6, FLAG-EFA6-EK, Arf6 Q67L, Arf1Q71L, FLAG- ARNO 2G, or FLAG-ARNO 3G with one or more PMA-induced ventral actin structure visible by phalloidin staining was quanti- fied and is expressed as the average percentage of transfected cells from three independent experiments with 100 cells scored in each experiment. Error bars represent one standard deviation from the mean. (D) Z-sections of HeLa cells, transfected with Flag-EFA6 that were untreated or treated with PMA for 15 min, fixed and stained with an antibody against Flag (green) and Rhodamine phal- loidin (red). A cross-section of the cell shows that Flag-EFA6, which associates with the plasma membrane, shows increased concen- tration on the ventral (bottom) surface upon stimulation with PMA, where the ventral actin structure creates a membrane fold, or ruffle. A blue line denotes the location of the cross-section. Bar, 10 lM.

Article Snippet: Western blots were carried out using a rabbit polyclonal antibody to Arf1, a rabbit polyclonal antibody to Arf6 (see details above) or a mouse monoclonal antibody to Arf6 (Santa Cruz Biotechnology), and a rabbit antibody to actin (Sigma), and a mouse monoclonal antibody to a-tubulin (DM1A) (Sigma).

Techniques: Transfection, Immunofluorescence, Staining, Labeling, Expressing, Standard Deviation, Clinical Proteomics, Membrane

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Arf1 and Arf6 promote ventral actin structures formed by acute activation of protein kinase C and Src.

doi: 10.1002/cm.21181

Figure Lengend Snippet: Fig. 6. Arf1 and Arf6 are required for ventral wave formation in Beas-2b cells. (A) Beas-2b cells were transfected with siRNA to knockdown Arf1 or Arf6, or mock treated (control), as described in the Materials and Methods section. 72 h after transfection, cells were fixed and stained by immunofluorescence as described in the Materials and Methods section with an anti- body against b-COP, and costained with rhodamine phalloidin. (B) Control and Arf1-or Arf6-depleted cells were treated with 200 nM PMA for 30 min prior to fixation and staining with rhodamine phalloidin. Bars, 10 mm. (C) The fraction of 200 cells with one or more visible ventral waves was quantified and is expressed as the average percentage from three independent experiments. Error bars represent one standard deviation of the mean. One-way ANOVA revealed that both siRNA-depletions differed from control (P < 0.01). (D) 5 3 105 cells were col- lected, lysed, and run on a SDS page gel, and immunoblotted with antibodies against Arf1, Arf6, and actin. Arf1 was depleted by 90% and Arf6 by 75%.

Article Snippet: Western blots were carried out using a rabbit polyclonal antibody to Arf1, a rabbit polyclonal antibody to Arf6 (see details above) or a mouse monoclonal antibody to Arf6 (Santa Cruz Biotechnology), and a rabbit antibody to actin (Sigma), and a mouse monoclonal antibody to a-tubulin (DM1A) (Sigma).

Techniques: Transfection, Knockdown, Control, Staining, Immunofluorescence, Standard Deviation, SDS Page

Journal: Journal of Extracellular Vesicles

Article Title: RAB22A sorts epithelial growth factor receptor (EGFR) from early endosomes to recycling endosomes for microvesicles release

doi: 10.1002/jev2.12494

Figure Lengend Snippet: RAB22A increases cell‐surface EGFR expression. (a, c) Representative Western blot showing cell surface EGFR protein level on HeLa cells treated with vehicle, 25 µM Dyngo‐4a (a), or 150 µM primaquine (c). (b, d) Representative Western blot showing EGFR level on MV from HeLa cells treated with vehicle, 25 µM Dyngo‐4a (b), or 150 µM primaquine (d). (e) Representative Western blot showing EGFR level on MV from HeLa cells expressing Vector, HA‐RAB11A, HA‐RAB11A Q70L or HA‐RAB11A S25N . (f) Representative Western blot showing cell surface EGFR level on HeLa cells expressing vector or FLAG‐RAB22A. (g) Representative Western blot showing cell surface EGFR level on HeLa cells with or without RAB22A knockout. (h–j) Cell surface EGFR level in A549 (h), NCI‐H1975 (i) and NCI‐H820 (j) cell lines which was transduced with control small interfering RNA (si#NC) or siRNA targeting RAB22A (si#1 and si#2) for 48 h was analysed by flow cytometry. Data represent mean ± s.e.m.; p < 0.05 was considered significant; two‐tailed unpaired t ‐test. FLOT2 was used as a loading control.

Article Snippet: Antibodies purchased from Proteintech Group: RAB22A Rabbit Polyclonal antibody (12125‐1‐AP, 1:500), ARF6 Rabbit Polyclonal antibody (20225‐1‐AP, 1:3000), Tsg101 Antibody (4497‐1‐AP, 1:3000), RAB11A‐Specific Polyclonal Antibody (20229‐1‐AP, 1: 500), RAB7A Antibody (55469‐1‐AP, 1:500).

Techniques: Expressing, Western Blot, Plasmid Preparation, Knock-Out, Transduction, Control, Small Interfering RNA, Flow Cytometry, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: RAB22A sorts epithelial growth factor receptor (EGFR) from early endosomes to recycling endosomes for microvesicles release

doi: 10.1002/jev2.12494

Figure Lengend Snippet: RAB22A engages SH3BP5L to activate RAB11A. (a) Localisation of FLAG‐RAB22A, EGFR‐HA and EGFP‐RAB11A in HeLa cells stably expressing FLAG‐RAB22A. Cells were co‐transfected with EGFR‐HA and EGFP‐RAB11A for 48 h. Pearson's correlation coefficients were calculated in the histogram, n = 30 cells. (b) Localisation of RAB22A, EGFR and RAB11A probed by anti‐RAB22A, anti‐EGFR and anti‐RAB11A antibodies respectively in NCI‐H1975 cells. Pearson's correlation coefficients were calculated in the histogram, n = 18 (EGFR/RAB11), 30 (RAB22A/RAB11) cells. (c) Whole cell lysate from HeLa cells stably expressing Vector or FLAG‐RAB22A was incubated with guanosine 5′‐triphosphate–agarose for 1.5 h, then the proteins were analysed by Western blot. (d) Whole cell lysate from HeLa cells with or without RAB22A knockout was incubated with guanosine 5′‐triphosphate–agarose for 1.5 h, then the proteins were analysed by Western blot. (e) HEK‐293T cells were transfected with the indicated plasmids. 48 h later, cells were lysed with RIPA and the lysate was incubated with anti‐HA agarose. Proteins were analysed by Western blot. (f) Localisation of HA‐SH3BP5 and HA‐SH3BP5L in HeLa cells stably expressing FLAG‐RAB22A. Cells were transiently transfected with HA‐SH3BP5 or HA‐SH3BP5L plasmid for 48 h. Pearson's correlation coefficients were calculated in the histogram, n = 32 (SH3BP5), 33 (SH3BP5L) cells. Data represent mean ± s.e.m.; p < 0.05 was considered significant; two‐tailed unpaired t ‐test. (g) Whole cell lysate from indicated cell lines were incubated with GST‐FIP3RBD‐coated beads and the proteins were analysed by Western blot. (h) HEK‐293T was transiently co‐transfected with FLAG‐RAB22A and indicated truncated mutants of SH3BP5L for 48 h. Cells were lysed with RIPA and the lysate was incubated with anti‐HA agarose. Proteins were analysed by Western blot.

Article Snippet: Antibodies purchased from Proteintech Group: RAB22A Rabbit Polyclonal antibody (12125‐1‐AP, 1:500), ARF6 Rabbit Polyclonal antibody (20225‐1‐AP, 1:3000), Tsg101 Antibody (4497‐1‐AP, 1:3000), RAB11A‐Specific Polyclonal Antibody (20229‐1‐AP, 1: 500), RAB7A Antibody (55469‐1‐AP, 1:500).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Knock-Out, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: RAB22A sorts epithelial growth factor receptor (EGFR) from early endosomes to recycling endosomes for microvesicles release

doi: 10.1002/jev2.12494

Figure Lengend Snippet: Tyr136 in RAB22A is phosphorylated by EGFR. (a) Representative Western blot of RAB22A tyrosine phosphorylation (pY) by EGFR and its mutants. HEK‐293T cells were transiently co‐transfected with the indicated plasmids, 42 h later, the medium was replaced with serum‐free DMEM and cells were cultured for another 6 h. (b) Localisation of EGFR pY1068 and EGFR T790M/L858R (EGFR M2 ‐HA) in HeLa cells stably expressing FLAG‐RAB22A. (c) Representative Western blot of tyrosine phosphorylation of RABB22A (pY) by EGFR M2 ‐HA in HEK‐293T. Cells were co‐transfected with the indicated plasmids. After 42 h, the medium was replaced with serum‐free DMEM in the absence and presence of 1 µM specific TKIs for another 6 h. (d) Representative Western blot to identify tyrosine in RAB22A phosphorylated by EGFR and its active mutants. HEK‐293T cells were co‐transfected with the indicated plasmids, 42 h later, the medium was replaced with serum‐free DMEM and cells were cultured for another 6 h. (e) In vitro kinase assay.

Article Snippet: Antibodies purchased from Proteintech Group: RAB22A Rabbit Polyclonal antibody (12125‐1‐AP, 1:500), ARF6 Rabbit Polyclonal antibody (20225‐1‐AP, 1:3000), Tsg101 Antibody (4497‐1‐AP, 1:3000), RAB11A‐Specific Polyclonal Antibody (20229‐1‐AP, 1: 500), RAB7A Antibody (55469‐1‐AP, 1:500).

Techniques: Western Blot, Phospho-proteomics, Transfection, Cell Culture, Stable Transfection, Expressing, In Vitro, Kinase Assay

Journal: Journal of Extracellular Vesicles

Article Title: RAB22A sorts epithelial growth factor receptor (EGFR) from early endosomes to recycling endosomes for microvesicles release

doi: 10.1002/jev2.12494

Figure Lengend Snippet: Phosphorylation of Tyr136 in RAB22A by EGFR promotes EGFR‐containing MV formation. (a) Representative Western blot of MV sample released by HeLa cells with RAB22A and wild‐type or constitutively active form of EGFR overexpression. (b) Representative Western blot of MV sample released by HeLa cells with RAB22A and wild‐type or kinase dead form of EGFR overexpression. (c) Representative Western blot of MV from HeLa cells in the presence or absence of 1 µM Afatinib for 48 h. (d) Representative Western blot of MV from HeLa cells stably expressing EGFR M2 ‐HA and V5‐RAB22A or its Y136F mutant. (e) Representative Western blot of MV from NCI‐H1975 cells with or without RAB22A knockout followed by RAB22A or its Y136F mutant re‐expression. FLOT2 was used as a loading control.

Article Snippet: Antibodies purchased from Proteintech Group: RAB22A Rabbit Polyclonal antibody (12125‐1‐AP, 1:500), ARF6 Rabbit Polyclonal antibody (20225‐1‐AP, 1:3000), Tsg101 Antibody (4497‐1‐AP, 1:3000), RAB11A‐Specific Polyclonal Antibody (20229‐1‐AP, 1: 500), RAB7A Antibody (55469‐1‐AP, 1:500).

Techniques: Phospho-proteomics, Western Blot, Over Expression, Stable Transfection, Expressing, Mutagenesis, Knock-Out, Control

Journal: Journal of Extracellular Vesicles

Article Title: RAB22A sorts epithelial growth factor receptor (EGFR) from early endosomes to recycling endosomes for microvesicles release

doi: 10.1002/jev2.12494

Figure Lengend Snippet: Proposed model by which RAB22A links the endocytosis and recycling pathway to promote MVs release. RAB22A recruits TBC1D2B to inactivate RAB7, and further prevents EGFR from being transported to late endosomes and later lysosomes for degradation, which constructs a reserve pool ready for recycling. RAB22A also engages SH3BP5L to promote RAB11A activation and increase cell‐surface EGFR protein level, which ultimately facilitates the release of EGFR‐containing MVs. Moreover, RAB22A can be phosphorylated by active EGFR, forming a positive feedback loop to promote the release of EGFR‐containing MVs.

Article Snippet: Antibodies purchased from Proteintech Group: RAB22A Rabbit Polyclonal antibody (12125‐1‐AP, 1:500), ARF6 Rabbit Polyclonal antibody (20225‐1‐AP, 1:3000), Tsg101 Antibody (4497‐1‐AP, 1:3000), RAB11A‐Specific Polyclonal Antibody (20229‐1‐AP, 1: 500), RAB7A Antibody (55469‐1‐AP, 1:500).

Techniques: Construct, Activation Assay

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.

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, 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.

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: 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:

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

Journal: PLoS ONE

Article Title: The Role of ARF6 in Biliary Atresia

doi: 10.1371/journal.pone.0138381

Figure Lengend Snippet: (A) Boxed region shows well stratified cases and controls carried forward in analysis. (B) The BA susceptibility locus defined by rs3126184 and rs10140366 lies in an enhancer region in the 3’ flanking region of the ARF6 gene (UCSC genome browser evaluation show enriched histone marks H3K4me1 overlayed with H3K27Ac is an indicative of enhancer region). (C) i-iv show ARF6 immunostaining in liver explants from normal children with intact bile ducts (BD) (i), children with BA with bile duct paucity in portal tracts (PT) (ii) or cirrhosis (iii), and a child with hepatocellular carcinoma (iv). T = tumor cells, L = lobule.

Article Snippet: Fourteen additional patients were genotyped with TaqMan® SNP Genotyping Assay (Life Technologies ID; C_11918520_10) for the candidate SNP; rs3126184.

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: The Role of ARF6 in Biliary Atresia

doi: 10.1371/journal.pone.0138381

Figure Lengend Snippet: (A, B) Whole-mount in situ hybridization showing arf6a and arf6b expression in developing embryos/larvae. Arrows point to the liver; arrowheads the pancreas; brackets mark the intestinal bulb. (C) The Tg(Tp1 : GFP) and Tg(fabp10a : dsRed) lines reveal the intrahepatic biliary structure and liver size, respectively. Epifluorescence images showing the expression of these transgenes revealed a defect in the intrahepatic biliary structure in arf6 -ATG MO-injected larvae. Based on the severity of the biliary defect, larvae were divided into three groups: normal, intermediate, and severe. Graph shows the percentage of larvae in each group. Arrows point to the liver; dotted lines outline the liver (A-C). (D) Epifluorescence images showing PED-6 accumulation in the gallbladder (arrows). Based on PED-6 levels in the gallbladder, larvae were divided into three groups: absent, small/faint, and normal. Graph shows the percentage of larvae in each group. n, the number of larvae examined; scale bars, 100 μm.

Article Snippet: Fourteen additional patients were genotyped with TaqMan® SNP Genotyping Assay (Life Technologies ID; C_11918520_10) for the candidate SNP; rs3126184.

Techniques: In Situ Hybridization, Expressing, Injection

Journal: PLoS ONE

Article Title: The Role of ARF6 in Biliary Atresia

doi: 10.1371/journal.pone.0138381

Figure Lengend Snippet: Instead of 4 μM AG1478 and 2 ng of arf6 -ATG MO, 1 μM AG1478 and 0.5 ng of arf6 -ATG MO were used. (A) Epifluorescence images showing the expression of Tp1 :GFP and fabp10a :dsRed revealed a severe defect in the intrahepatic biliary structure only when the MO injection was combined with the AG1478 treatment. Based on the severity of the biliary defect, larvae were divided into three groups: normal, intermediate, and severe. Arrows point to the liver and dotted lines outline the liver. Scale bars, 100 μm. (B) Graph showing the percentage of larvae in each group shown in A. (C) Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder at 5 dpf. n indicates the number of larvae examined.

Article Snippet: Fourteen additional patients were genotyped with TaqMan® SNP Genotyping Assay (Life Technologies ID; C_11918520_10) for the candidate SNP; rs3126184.

Techniques: Expressing, Injection

Journal: PLoS ONE

Article Title: The Role of ARF6 in Biliary Atresia

doi: 10.1371/journal.pone.0138381

Figure Lengend Snippet: The relative expression level of genes was shown in fold change in arf6 morphants over un-injected controls. Error bars shown, ± SEM (average of three independent experiments).

Article Snippet: Fourteen additional patients were genotyped with TaqMan® SNP Genotyping Assay (Life Technologies ID; C_11918520_10) for the candidate SNP; rs3126184.

Techniques: Expressing, Injection

Journal: PLoS ONE

Article Title: The Role of ARF6 in Biliary Atresia

doi: 10.1371/journal.pone.0138381

Figure Lengend Snippet: Ligation of activated EGFR to GEP100 initiates sequential activation of ARF6, ERK/MAPK and CREB signaling proteins resulting in cellular development and proliferation. Negative regulation of ARF6 originating from the regulatory regions defined by rs3126184 and rs10140366 in human BA, and arf6 knockdown or EGFR inhibition with AG1478 in zebrafish embryos lead to poor bile duct development.

Article Snippet: Fourteen additional patients were genotyped with TaqMan® SNP Genotyping Assay (Life Technologies ID; C_11918520_10) for the candidate SNP; rs3126184.

Techniques: Ligation, Activation Assay, Knockdown, Inhibition

Journal: Journal of Molecular Cell Biology

Article Title: Acetylation of ACAP4 regulates CCL18-elicited breast cancer cell migration and invasion

doi: 10.1093/jmcb/mjy058

Figure Lengend Snippet: ACAP4 is required for CCL18-elicited breast cancer cell migration. (A) ARF6 and ACAP4 distribution profiles in the MDA-MB-231 cells. Breast cancer cells were starved from serum for 6 h before stimulated with 20 ng/ml CCL18 for 10 min. Cells were fixed, permeabilized, and stained for endogenous ARF6 (green), ACAP4 (red), and DAPI (blue). The merged montage was generated from three channels. Scale bar, 10 μm. (B) Quantitative analyses for the effect of ACAP4 on ARF6-dependent formation of protrusions. MDA-MB-231 cells were treated with scramble or ACAP4 siRNA for 24 h followed by CCL18 stimulation (20 ng/ml) for 10 min prior to fixation. The data are presented as the fraction of cells forming ARF6-rich protrusions normalized to the fraction of scramble siRNA-treated cells stimulated with CCL18. The error bars represent SEM; n = 3 preparations. (C) MDA-MB-231 cells were transfected with the ACAP4 siRNA oligonucleotides for 24 h and subjected to SDS-PAGE and immunoblotting. Top panel, immunoblot for ACAP4; middle panel, immunoblot for ezrin; bottom panel, immunoblot for ARF6. Scrambled oligonucleotides were used as controls. (D) Depletion of ACAP4 inhibits wound-healing cell migration. MDA-MB-231 cells treated with siRNA against ACAP4 or a scrambled control were examined in the wound-healing assay. Images were collected before or 4 and 8 h after the CCL18 addition (20 ng/ml). Results are representative of three independent experiments. (E) Quantitative analyses of wound-healing cell migration in D. The number of migrating cells depleted of ACAP4 to the wound area was compared with that of scrambled siRNA-treated MDA-MB-231 cells and then expressed as a percentage. The mean with SEM was then derived from three independent experiments. NS, no significant difference; **P < 0.01.

Article Snippet: Anti-HA antibody, anti-Myc antibody, anti-ARF6 antibody and anti-acetylated-lysine antibody were purchased from Cell Signaling Technology.

Techniques: Migration, Staining, Generated, Transfection, SDS Page, Western Blot, Control, Wound Healing Assay, Derivative Assay

Journal: Journal of Molecular Cell Biology

Article Title: Acetylation of ACAP4 regulates CCL18-elicited breast cancer cell migration and invasion

doi: 10.1093/jmcb/mjy058

Figure Lengend Snippet: CCL18-elicited acetylation promotes ARF6 GTPase activity via dissociation of ACAP4 from the plasma membrane. (A) Co-immunoprecipitation of Myc-ARF6 and FLAG-ACAP4 wild-type or variants. 293T cells were transfected with Myc-ARF6 plus FLAG-ACAP4 wild-type (WT) or variants (K311Q/R). Twenty-four hours after transfection, cells were lysed and incubated with the FLAG M2 beads for 2 h. Then the beads were washed and analyzed by western blotting with anti-Myc and anti-FLAG antibodies. (B) Endogenous ARF6 activity was measured by GGA3 pull-down assay with CCL18 simulation. MDA-MB-231 cells were subjected to serum starvation followed by stimulation with 20 ng/ml CCL18 for 10 min or 0.1 μg/ml EGF for 5 min. The stimulated cells were incubated with the GST-GGA3GAT. Active forms of ARF6 were measured by anti-ARF6-GTP blot. (C) Quantitative analyses on the ratio of ARF6-GTP/ARF6-ALL described in B. Data represent mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. (D) MDA-MB-231 cells were transfected with ACAP4-GFP wild-type (WT) or acetylation-mimicking variants (K311Q/R). At 16 h after transfection, cells were subjected to CCL18 stimulation. Treated cells were fixed and then examined under fluorescence microscope. Note that persistent acetylation mutant or CCL18 stimulation attenuated the membrane-associated ACAP4 localization. Scale bar, 10 μm. (E) Quantitative analyses of membrane protrusion rich in ARF6. Data represent mean ± SEM from three independent experiments. **P < 0.01. (F) CCL18 stimulation liberates ACAP4 from plasma membrane association. MDA-MB-231 cells were transfected with ACAP4-GFP wild-type (WT) or acetylation-mimicking variants (K311Q/R). At 16 h after transfection, cells were subjected to CCL18 stimulation followed by digitonin extraction to separate cytosol from membrane fraction. (G) Quantitative analyses of ACAP4 partitioning in membrane and cytosol. Data represent mean ± SEM from three independent experiments. *P < 0.05.

Article Snippet: Anti-HA antibody, anti-Myc antibody, anti-ARF6 antibody and anti-acetylated-lysine antibody were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Clinical Proteomics, Membrane, Immunoprecipitation, Transfection, Incubation, Western Blot, Pull Down Assay, Fluorescence, Microscopy, Mutagenesis, Extraction

Journal: Journal of Molecular Cell Biology

Article Title: Acetylation of ACAP4 regulates CCL18-elicited breast cancer cell migration and invasion

doi: 10.1093/jmcb/mjy058

Figure Lengend Snippet: Acetylation of K311 orchestrates CCL18-elicited breast cancer cell migration and invasion. (A) Western blotting analyses of exogenous expression of GFP-ACAP4 wild-type and K311Q/R mutants in MDA-MB-231 cells. (B) Acetylation of Lys311 is necessary in MDA-MB-231 cell migration. MDA-MB-231 cells were transfected with WT or the K311Q/R mutants of ACAP4. Wound-healing assay was performed as in Figure ​Figure1D1D and the migrated cells were counted. Quantitative analyses of migration ratio are shown. Data represent mean ± SEM. NS, no significant difference; *P < 0.05; **P < 0.01; n = 3. (C) Schematic drawing of cell migration trajectory. The total distance between the starting and ending points (T) and the actual trajectory (D) are indicated. (D) Quantitative analyses of directional distance. Data represent mean ± SEM. n = 31 and 29, respectively. *P < 0.05; **P < 0.01. (E) PCAF–ACAP4 signaling is essential for MDA-MB-231 cell invasion. MDA-MB-231 cells were transfected with ACAP4 WT or K311Q/R mutants and set to the Boyden chamber assay. Invasive cells through the membrane were stained and imaged. Note that the cells passing through the Boyden chamber were indicated by arrows. (F) Quantitative analyses of invasion efficiency shown in E. Data represent mean ± SEM. **P < 0.01; n = 3. (G) Working model. In breast cancer cells, CCL18 binds to its receptor on the plasma membrane of breast cancer cells and transacts PCAF activity. PCAF-acetylated ACAP4 binds to the plasma membrane and induces the release of ACAP4 to the cytosol. Meanwhile, ARF6 is recruited to the plasma membrane by the CCL18 signal; the spatial separation of ACAP4 from ARF6 at the plasma membrane enhances the ARF6 GTPase activity. We propose that the dynamic cycling of ACAP4 between membrane and cytosol accounts for the drive behind ARF6- and ACAP4-dependent cell migration and invasion.

Article Snippet: Anti-HA antibody, anti-Myc antibody, anti-ARF6 antibody and anti-acetylated-lysine antibody were purchased from Cell Signaling Technology.

Techniques: Migration, Western Blot, Expressing, Transfection, Wound Healing Assay, Boyden Chamber Assay, Membrane, Staining, Clinical Proteomics, Activity Assay