Journal: Scientific Reports

Article Title: Unique Lipid Signatures of Extracellular Vesicles from the Airways of Asthmatics

doi: 10.1038/s41598-018-28655-9

Figure Lengend Snippet: Heterogeneity of isolated BAL EVs. ( a ) The proportions of EVs which are CD9 + CD63 + CD81 + TSG101 + . Mann Whitney T test, ** < 0.01. ( b ) The proportions of ARF6 − , GRP94 − , and ARF6 − GRP94 − EVs. ( c ) The proportions of ARF6 − , GRP94 − , and ARF6 − GRP94 − EVs that are CD9 + CD63 + CD81 − TSG101 − . ( d ) The proportions of ARF6 − , GRP94 − , and ARF6 − GRP94 − EVs that are CD9 − CD63 − CD81 + TSG101 + . Mann Whitney T test, ** < 0.01. (Healthy Subjects, n = 8; Asthmatic Subjects, n = 7).

Article Snippet: EVs were also stained with antibodies to HLA-DR APC (clone LN3; Affymetrix, Inc., Santa Clara), CD54 PE (clone HA58; Affymetrix, Inc., Santa Clara), CD9 PE (clone M-L13; BD Biosciences, San Jose, CA), CD81 PE-Cy7 (clone 5A6; BioLegend, Inc., San Diego, CA), Grp94 DyLight 488 (clone 9G10; Enzo Life Sciences, Inc., Framingdale, NY), and ARF6 APC (AssayPro, LLC, St. Charles, MO).

Techniques: Isolation, MANN-WHITNEY

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:

Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

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

Journal: PLoS ONE

Article Title: The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

doi: 10.1371/journal.pone.0018458

Figure Lengend Snippet: A. Transient over-expression of ARF6(Q67L) in GFP-GAP273(3.2) promotes cell spreading (left panels). Actin foci are also absent from the cell. Transient over-expression of ARF6(T27N) in GFP-GAP273(3.2) cells (right panels) promotes or stabilizes the formation of actin foci. Images of GFP-GAP273 fluorescence of the same cells are shown in the bottom panels. The GFP-GAP273 fluorescence co-localizes with ARF6 staining in the cell transfected with ARF6(T27N). B. Transient over-expression of Rac1(G12V) (top left) promotes cell spreading and induces membrane ruffling in GFP-GAP273(3.2) cells but suppresses the localization of peripheral concentrations of GFP-GAP273 at the actin foci (compare the cell transfected with Rac1(G12V) with the surrounding non-transfected cells, bottom left). Transient over-expression of Rac1(T17N) (top right) in GFP-GAP273(3.2) cells inhibits cell spreading and prolongs the appearance of membrane projections containing GFP-GAP273. The GFP image (bottom right) shows that surrounding non-transfected cells are more spread and contain little, if any, peripheral concentrations of GFP-GAP273. For cells transfected with Rac1(G12V) or ARF6(Q67L), the cells had been re-plated on glass cover slips for about 20 hours before fixation and immunofluorescence labeling. For cells transfected with Rac1(T17N) or ARF6(T27N), the cells had been re-plated for 40 hours. Scale bar = 25 µm. C. Quantification of the effect of Rac1(T17N) and ARF6(T27N) on the formation of actin foci. The percentage of cells transiently over-expressing Rac1(T17N) or ARF6(T27N) in GFP-GAP273(3.2) cells was measured. In each of the three independent experiments, one hundred cells were counted. Error bar = S.D. D. Quantification of the effect of Rac1(G12V) and ARF6(Q67L) on cell spreading. The area covered by GFP-GAP273(3.2) cells transiently over-expressed with Rac1(G12V) or ARF6(Q67L) was measured. The mean area covered were obtained from 41, 60 and 123 cells, for ARF6(Q67L), Rac1(G12V) or non-transfected GFP-GAP273(3.2), respectively. Error bar = S.D.

Article Snippet: Goat anti-rabbit IgG (for ARF6) and goat anti-mouse IgG (for Rac1) secondary antibodies (Invitrogen) conjugated with Alexa fluorophores were used to generate fluorescence signals.

Techniques: Over Expression, Fluorescence, Staining, Transfection, Immunofluorescence, Labeling, Expressing

Journal: PLoS ONE

Article Title: The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

doi: 10.1371/journal.pone.0018458

Figure Lengend Snippet: A. Activation of Rac1 is inhibited in CHO cells stably expressing GFP-GAP273, but not in wild type CHO cells. The result shown is a representative of three independent experiments. B. GFP-GAP273 did not inhibit the activation of RhoA. C. Serum caused little change in binding of GTP by ARF6 in either cell type and GFP-GAP273 did not affect ARF6 activation. The percent of total GTP-binding protein that was bound to GTP in each sample is listed below the image of the immunoblots.

Article Snippet: Goat anti-rabbit IgG (for ARF6) and goat anti-mouse IgG (for Rac1) secondary antibodies (Invitrogen) conjugated with Alexa fluorophores were used to generate fluorescence signals.

Techniques: Activation Assay, Stable Transfection, Expressing, Binding Assay, Western Blot

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: ARF1 and ARF6 knock down impairs HGF-stimulated recycling of GRASP and Dock180. (A-C) MDCK cells were transfected with control siRNA or siRNA against either ARF6 (B) or ARF1 (C) by Neon transfection and plated on fibronectin-coated coverslips as described in the Materials and Methods. The following day, cells were transfected with GRASP and Dock180 by Lipofectamine 3000 and allowed to express for 10–12 hours. Cell were switched to serum-free media overnight and treated with HGF (10 ng/mL) the next morning. Cells were fixed at the indicated time points and imaged by deconvolution microscopy. Scale bars: 10 um. (D, E) Slidebook 6.0 imaging software was used to calculate levels of Dock180 (D) and GRASP (E) at the cell periphery in 62–78 cells as described in the Materials and Methods. Data are means ± standard error of the peripheral sum intensity normalized to the whole cell sum intensity. Asterisk indicates statistically significant compared to zero hour of set. * = p < 5 × 10−5, T test.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Transfection, Microscopy, Imaging, Software

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: Inhibition of ARF-associated recycling pathways impairs HGF-stimulated Rac activation. (A, B) Expression of Rab8 T22N or Rab11 S25N impairs HGF-stimulated Rac activation. MDCK cells were transfected with mTurquoise 2-Rab8 T22N, Rab11 S25N, or empty vector using Lipofectamine 3000. Cells were allowed to express for 10 hours and switched to serum-free media overnight. Cells were incubated in the presence or absence of HGF (20ng/mL) and lysed after 6 hours. GST-PBD pull downs were performed to isolate Rac-GTP. Starting lysate and pull down samples were run on a Western and blotted for Rac and GFP (Rabs). Representative gels are shown. Experiments were quantified by the LI-COR imaging system; Rab8 T22N (A, n = 8) and Rab11 S25N (B, n=7). Data shown are means ± standard error. * = p < 0.05, paired T test. (C, D) ARF1 and ARF6 knockdowns impair HGF-mediated Rac activation. MDCK cells were transfected with control siRNA or siRNA against ARF1 or ARF6 by Neon transfection. After 30 hours of expression, cells were switched to serum-free media overnight. The next day, cells were incubated in the absence or presence of 20 ng/mL HGF. After 6 hours, cells were lysed and Rac-GTP was isolated by GST-PBD pull down as described above. ARF6 (C) n = 7, ARF1 (D) n = 9. Data shown are means ± standard error. * = p < 0.05, paired T test.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Inhibition, Activation Assay, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Imaging, Isolation

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: Inhibition of ARF trafficking pathways traps GRASP and Dock180 together. (A-C) Expression of Rab8 T22N and Rab11 S25N traps GRASP and Dock180 in resting cells. MDCK cells were plated on fibronectin-coated coverslips and transfected with YPET-GRASP, mCherry-Dock180, and either mTurquoise 2-Rab8 T22N, Rab11 S25N or empty vector by Lipofectamine 3000. Cells were allowed to express for 10–12 hours, serum-starved overnight, and treated with HGF (10 ng/mL) the following morning. Cell were fixed and imaged by deconvolution microscopy as described in the Materials and Methods. (D) Extended inhibition of cytohesin GEF activity promotes GRASP and Dock180 binding. Following transfection and 10–12 hours of expression as described previously, cells were switched to serum-free media and treated with SecinH3 (15 uM). The next morning, cells were treated with HGF (10 ng/mL) and fixed at the designated time points. Scale bar: 10 um. (E-G) Knockdown of ARF1 and ARF6 promotes GRASP and Dock180 interaction. MDCK cells were Neon transfected with control siRNA or siRNA against either ARF1 or ARF6 and plated on fibronectin-coated coverslips. The next day, cells were transfected with YPET-GRASP and mCherry-Dock180 by Lipofectamine 3000, allowed to express for 10–12 hours, and switched to serum-free media overnight. Cells were treated with HGF (10 ng/mL) the next morning and fixed at the indicated time points. Scale bar: 10 um. (H) Quantification of corrected FRET signal by Slidebook 6.0 in 67–70 cells expressing either Rab8 T22N, Rab11 S22N or treated with SecinH3. Data are means ± standard error, * = p < 5 × 10−4, ** p < 5 × 10−7, *** p < 5 × 10−20, T test. Corrected FRET intensity values in scale bar are in arbitrary units. (I) Whole cell FRET sum intensity was calculated using Slidebook 6.0 in 62–78 cells. Data are means ± standard error. Asterisk indicates statistically significant compared to zero hour of set. * p < 5 × 10−9, T test. Corrected FRET intensity values in scale bar are in arbitrary units.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Microscopy, Activity Assay, Binding Assay

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: Trafficking of GRASP and Dock180 during ARF6 to Rac cross talk. GRASP and Dock180 localize to recycling endosomes in resting cells. HGF stimulation promotes association of GRASP and Dock180 and trafficking to the plasma membrane. Movement to the plasma membrane positions Dock180 to activate the membrane-localized Rac1. Inhibition of ARF6-associated trafficking pathways blocks HGF-stimulated movement of GRASP and Dock180 to the periphery and Rac1 activation. Inhibition of trafficking also traps GRASP and Dock180 together in the cell.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Inhibition, Activation Assay