Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 4. Rab35 and centaurin-b2 colocalize with Arf6. (A) Subcellular localization of endogenous Rab35 and Arf6 after NGF stimulation for 6 hours. NGF-stimulated PC12 cells were fixed, and then stained with anti-Rab35 antibody (green; 1:100 dilution), anti-Arf6 antibody (red; 1:100 dilution), and DAPI (nuclei; blue). Note that Rab35 and Arf6 are colocalized in the perinuclear area (arrowheads). Scale bar: 10 mm. The arrowheads indicate colocalization between Rab35 and Arf6. (B) Subcellular localization of endogenous centaurin-b2 (Centb2) and Arf6 after NGF stimulation. After NGF stimulation for 6 hours PC12 cells were fixed, and then stained with anti- centaurin-b2 antibody (green; 1:100 dilution), anti-Arf6 antibody (red; 1:100 dilution) and DAPI (nuclei; blue). Note that centaurin-b2 and Arf6 are colocalized in the perinuclear area (arrowheads). Scale bar: 10 mm. The lower panels in A and B are magnified views of the boxed areas in the upper right panels. The arrowheads indicate colocalization between centaurin-b2 and Arf6. (C) Intensity scatterplot of Rab35 signals (green) and Arf6 signals (red) in PC12 cells after NGF stimulation for 6 hours. The Pearson’s correlation coefficient (PCC) (mean and s.d.) for the relation between them is shown at the bottom (n530 from three independent experiments). (D) Intensity scatterplot of centaurin-b2 signals (green) and Arf6 signals (red) in PC12 cells after NGF stimulation for 6 hours. The PCC (mean and s.d.) for the relation between them is shown at the bottom (n530 from three independent experiments).

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Staining

Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 5. Arf6-GAP activity of centaurin-b2 is required for NGF-induced neurite outgrowth of PC12 cells after Rab35-dependent recruitment. (A) Colocalization analysis of wild-type centaurin-b2 (Centb2-WT), centaurin-b2(DANKR) (Centb2-DANKR), and centaurin-b2(RQ) (Centb2- RQ) with endogenous Arf6 in PC12 cells after NGF stimulation for 36 hours. PC12 cells transiently expressing either Myc-Centb2(WT), Myc- Centb2(DANKR), or Myc-Centb2(RQ) (left panels) were fixed, and then stained with anti-Myc antibody (left panels; 1:500 dilution) and anti-Arf6 antibody (middle panels; 1:100 dilution). Merged images are shown in the right panels. Fluorescence intensity along white dashed lines (right panels) is shown on the right. Note that the Rab35-binding-deficient Centb2(DANKR) mutant had impaired ability to localize at the Arf6-positive pericentrosomal compartment. Scale bar: 5 mm. (B) Typical images of EGFP–Centb2(WT)SR- expressing and EGFP–Centb2(DANKR)SR-expressing PC12 cells after NGF stimulation for 36 hours under centaurin-b2-depleted conditions. Scale bar: 30 mm. (C) Effect of EGFP–Centb2(WT)SR and EGFP–Centb2(DANKR)SR

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Activity Assay, Expressing, Staining, Fluorescence, Binding Assay, Mutagenesis

Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 7. A model of the crosstalk between Rab35 and Arf6 through centaurin-b2 during neurite outgrowth. Rab35 is recruited to the pericentrosomal Arf6-positive endosomes in response to NGF stimulation and recruits its effector, centaurin-b2, to the same compartment in PC12 cells (Figs 2–4). Centaurin-b2 then inactivates Arf6 at the pericentrosomal endosomes through its Arf6-GAP activity during neurite outgrowth (Fig. 5). Inactivation of Arf6 at the pericentrosomal endosomes is required for the Rab35-mediated neurite outgrowth (Figs 1,6).

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Activity Assay

Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 6. Arf6 functions downstream of Rab35 in neurite outgrowth of PC12 cells. (A) Typical images of EGFP–Rab35(QL) + control siRNA (siControl)-expressing and EGFP–Rab35(QL) + Arf6 siRNA (siArf6)- expressing PC12 cells after NGF stimulation for 36 hours. Scale bar: 30 mm. (B) Typical images of EGFP–Rab35(QL) + mStr-expressing and EGFP– Rab35–(QL) + Arf6(QL)–mStr-expressing PC12 cells after NGF stimulation for 36 hours. Scale bar: 30 mm. (C) Effect of Arf6 knockdown on active Rab35-promoted neurite outgrowth of PC12 cells. Bars represent the total neurite length values (mean and s.e.) of EGFP + siControl-expressing (control; black bar), EGFP + siArf6-expressing (left white bar), EGFP– Rab35(QL) + siControl-expressing (central white bar) and EGFP–Rab35(QL) + siArf6-expressing (right white bar) cells (n.100). **P,0.01, in comparison with the control cells (Student’s unpaired t-test). Note that Rab35(QL)-dependent promotion of neurite outgrowth was dramatically inhibited by knockdown of endogenous Arf6. (D) Effect of expression of Arf6(QL) on active Rab35-promoted neurite outgrowth of PC12 cells. Bars represent the total neurite length values (mean and s.e.) of EGFP + mStr- expressing (control; black bar), Arf6(QL)–mStr + EGFP-expressing (left white bar), mStr + EGFP–Rab35(QL)-expressing (central white bar) and Arf6(QL)–mStr + EGFP–Rab35(QL)-expressing (right white bar) cells (n.100). **P,0.01, in comparison with the control cells (Student’s unpaired t-test). Note that the Rab35(QL)-dependent promotion of neurite outgrowth was completely inhibited by co-expression with Arf6(QL).

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Control, Expressing, Knockdown, Comparison

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: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A) STZ-induced diabetic retinal permeability in endothelium-driven Arf6-KO mice compared with that observed in littermate controls. (B) Glucose levels of mice used for A. (C) VEGF-induced retinal permeability in Arf6–conditional KO mice compared with that observed in littermate controls. Error bars represent the SEM. In A (n ≥8) and C (n ≥6), statistical significance was determined by Welch’s 1-way ANOVA and Games-Howell multiple comparisons test for unequal variances.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Permeability

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A) ARF6 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2 phosphorylation. (B) ARF6 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for PLCγ, p38, ERK1/2, and MARCKS phosphorylation. (C) HRMECs infected with Ad-null, Ad-ARF6WT, or Ad-ARF6Q67L were cultured in endothelial growth media and assayed for VEGFR2 phosphorylation. (D) ARF6 siRNA–treated HRMECs were assayed for VEGF-induced migration. In C (n = 3), geometric means and 95% CIs (error bars) of the ratios (each data point was normalized to its respective Ad-null control) were plotted on a logarithmic scale. Statistical significance was assessed using the ratio paired, 2-tailed t test on non-normalized data, and the P values are shown in the graph. (D) *P < 0.05, by 1-way ANOVA with Tukey’s multiple comparisons test (n = 6). All error bars represent the SEM. p, phosphorylated.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Control, Phospho-proteomics, Infection, Cell Culture, Migration

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A and B) ARF6 siRNA– or control siRNA–treated HRMECs were labeled with biotin, stimulated with VEGF for 5 minutes, and assayed for internalized VEGFR2. IB, immunoblot. (C) ARF6 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for colocalization of internalized VEGFR2 with endosome markers. Scale bar: 30 μm; original magnification, ×1,200. The images in C show a single optical confocal section through an internal region of the HRMECs; the Z-stacked images are shown in Supplemental Figure 3B. (D) Pitstop 2–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2, ERK1/2, and MARCKs phosphorylation. In B (n = 5), geometric means and 95% CIs (error bars) of the ratios (each data point normalized was to its respective untreated control) were calculated, and the ratios were plotted on a logarithmic scale. Statistical significance was assessed using the ratio paired, 2-tailed t test on non-normalized data, and the P value is shown in each graph.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Control, Labeling, Western Blot, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A) VEGF-treated HRMECs were assayed for ARF6 activation using an ARF6-GTP–pulldown assay. (B) ARNO siRNA–treated HRMECs were assayed for VEGF-induced ARF6 activation. (C) ARNO siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2 phosphorylation. (D) ARNO siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for PLCγ, p38, ERK1/2, and MARCKS phosphorylation. (E) ARNO siRNA– or control siRNA–treated HRMECs were labeled with biotin, stimulated with VEGF for 5 minutes, and assayed for internalized VEGFR2. (F) ARNO siRNA– or control siRNA–treated HRMECs were assayed for VEGF-induced cell migration. (G) HRMECs infected with Ad-null, Ad-ARNOWT, or Ad-ARNOE156K were stimulated with VEGF and assayed for VEGFR2 phosphorylation. In A (n = 5) and E (n = 5), geometric means and 95%CIs (error bars) of the ratios (each data point was normalized to its respective untreated control) were calculated, and the ratios were plotted on a logarithmic scale. Statistical significance was assessed using the ratio paired, 2-tailed t test on non-normalized data, and the P value is shown in each graph. In B (n = 3), F (n = 4), and G (n = 3), a 1-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001). All error bars represent the SEM.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Activation Assay, Control, Phospho-proteomics, Labeling, Migration, Infection

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A) GEP100 siRNA– or control siRNA–treated HRMECs were assayed for VEGF-induced ARF6 activation. (B) GEP100 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2, ERK1/2, and MARCKS phosphorylation. A representative blot from at least 4 independent experiments is shown. (C) GEP100 siRNA– or control siRNA–treated HRMECs were assayed for VEGF-induced cell migration. (D) GEP100 siRNA– or control siRNA–treated HRMECs were labeled with biotin, stimulated with VEGF for 5 minutes, and assayed for internalized VEGFR2. (E) ARF6 siRNA–, ARNO siRNA–, GEP100 siRNA–, or control siRNA–treated HRMECs were stimulated with VEGF for 60 minutes and assayed for VEGFR2 levels. (F) ARF6 siRNA–, ARNO siRNA–, GEP100 siRNA–, or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for colocalization of VEGFR2 with the lysosomal marker LAMP1. Scale bars: 30 μm; original magnification, ×1,200 and additional ×7 (inset). The images in F show a single optical confocal section through an internal region of the HRMECs, and the Z-stacked images are shown in Supplemental Figure 4K. In D, the geometric means and 95% CIs (error bars) of the ratios (each data point was normalized to its respective untreated control) were calculated, and the ratios were plotted on a logarithmic scale. Statistical significance was assessed using the ratio paired, 2-tailed t test, and the P value is shown in each graph. In A (n = 3), C (n = 4), and E (n = 3), a 1-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance for each time point (**P < 0.01). All error bars represent the SEM.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Control, Activation Assay, Phospho-proteomics, Migration, Labeling, Marker

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A) ARF6 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes, VEGFR2 was immunoprecipitated, and the precipitate was immunoblotted using antibodies against NRP1. Groupings of images are different parts of the same gel. (B) ARNO siRNA–, GEP100 siRNA–, or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2-NRP1 coimmunoprecipitation as described for A. (C) GEP100 siRNA– or control siRNA-treated HRMECs were infected with AdARF6Q67L for 36 hours and then stimulated with VEGF for 5 minutes and assayed for VEGFR2-NRP1 coimmunoprecipitation as described for A. (D) Pitstop 2–treated and/or GEP100 siRNA– or control siRNA–transfected HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2/NRP1 coimmunoprecipitation as described for A. (E) HRMECs were stimulated with VEGF for 5 minutes and assayed by immunofluorescence to determine the intracellular location of ARNO and GEP100. Scale bars: 30 μm; original magnification, ×1,200. In A–D, a 1-way ANOVA with Tukey’s multiple comparisons tests was used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). All error bars represent the SEM. n = 3 for all panels.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Control, Immunoprecipitation, Infection, Transfection, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: (A–C) Effects of NAV-2729 on VEGF-induced ARF6-GTP levels (A), VEGFR2 phosphorylation (B), and migration (C) of HRMECs. (D) STZ-induced diabetic permeability in mice treated with DMSO, SU4312, or NAV-2729. (E) STZ-induced diabetic neovascularization in rats treated with DMSO or NAV-2729. (F) STZ-induced diabetic pinpoint leakage in rats treated with DMSO or NAV-2729. (G) STZ-induced diabetic optic disc hyperfluorescence in rats treated with DMSO or NAV-2729. Dotted lines between data points represent the same animal after different treatments. In D (n ≥18), statistical significance was determined by Welch’s 1-way ANOVA and Games-Howell multiple comparisons test for unequal variances. In E–G (n ≥11), a paired, 2-tailed t test was used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). All error bars represent the SEM.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Phospho-proteomics, Migration, Permeability

Journal: The Journal of Clinical Investigation

Article Title: Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

doi: 10.1172/JCI91770

Figure Lengend Snippet: VEGF stimulation induces VEGFR2 dimerization and phosphorylation as well as ARF6 activation. The effect of ARF6 on this pathway is specified by the particular GEF that activates it. GEP100-dependent ARF6 activity promotes VEGFR2 binding to its coreceptor NRP1 at the cell surface, while ARNO-dependent ARF6 activity promotes VEGFR2 internalization. When bound to NRP1, VEGFR2 exhibits decreased lysosomal localization and degradation and increased plasma membrane recycling. Thus, ARF6 mediates VEGFR2 internalization and coreceptor binding, 2 trafficking mechanisms that are necessary for maximal VEGFR2 phosphorylation and downstream signaling. Note: phosphorylation at Y1175 has been shown to increase upon internalization of VEGFR2 (41). For simplicity, we illustrate this increase by showing only 1 of the 2 VEGFR2 chains phosphorylated at the plasma membrane. However, the true phosphorylation state of each chain at the plasma membrane has not yet been elucidated.

Article Snippet: Mouse anti-ARF6 antibody was from MilliporeSigma or Cell Biolabs, mouse anti-VEGFR2 was from R&D Systems, goat anti-NRP1 antibody was from Santa Cruz Biotechnology, and rabbit anti-GEP100 antibodies were from MilliporeSigma ( Supplemental Tables 4 and 5 ). siRNAs were purchased from QIAGEN ( Supplemental Table 6 ).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay, Binding Assay, Clinical Proteomics, Membrane

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: Cytohesins are required to promote HGF-induced activation of Rac1 and Arf6 in injured kidney. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and stained either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Staining

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Incubation, Staining

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: HGF induces the activation of Rac1 and Arf6 in damaged kidneys early after reperfusion. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both 24 h after reperfusion. Injured kidneys were then harvested 24 h after treatment, cryosectioned (10 mm), and stained either against GTP-Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Staining

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: Cytohesin-dependent Arf6 to Rac1 signaling module is required to promote the HGF-stimulated recovery of damaged kidney. Treatment of IRI kidneys with HGF activates the HGF receptor c-met, resulting in a signaling cascade event that leads to activation of the Arf GEFs cytohesins. Active cytohesins promote Arf6 to exchange its bound GDP molecule for GTP, resulting in its activation. GTP-bound Arf6 thus activates Rac1. Activation of Rac1 induces epithelial cell migration to the bare areas of the tubules, promoting damaged kidney recovery.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Migration