Journal: Journal of Biological Chemistry

Article Title: A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

doi: 10.1074/jbc.m404196200

Figure Lengend Snippet: FIG. 1. Binding of AMAP2 with GTP-Arf6. A and B, 5 g of GST-ArfGAP (A), GST-GGA3 (B), or GST alone, each produced in E. coli and purified on glutathione-beads, were incubated with 250 g of COS-7 cell lysates expressing various HA-Arfs, as indicated, and proteins co-precipitated with the GST fusion proteins were analyzed by immunoblotting. C, COS-7 cells were transfected with pcDNA-Arf6-HA and radiolabeled with [32P]orthophosphate. Cell lysates were then incubated with 5 g each of GST-ArfGAP (lane 1) or GST alone (lane 2), and bound radioactivities were analyzed by thin layer chromatography, as described under “Experimental Procedures.” As a control, HA-Arf6 was immuno- precipitated using an anti-HA antibody and similarly analyzed (lane 3). A representative result is shown from four independent experiments. D, 0.3 g of pcDNA-Arf-HA was co-transfected with 4 g of pEBG-AMAP2 or with 2 g of pEBG vector (for GST alone) plus 2 g of pcDNA empty vector in COS-7 cells, as indicated. GST proteins were then pulled down from 500 g of the cell lysates, prepared in 1% Nonidet P-40 buffer in the absence or presence of 10 mM Mg2, and analyzed for the co-precipitation of Arf isoforms. In A, B, and D, tagged proteins were detected by antibodies against tags, as indicated. The total includes 20 g of total cell lysate.

Article Snippet: Mouse monoclonal anti-Arf6 antibody and rabbit polyclonal anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Binding Assay, Produced, Purification, Incubation, Expressing, Western Blot, Transfection, Thin Layer Chromatography, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

doi: 10.1074/jbc.m404196200

Figure Lengend Snippet: FIG. 3. Subcellular localization of AMAP2 in HeLa cells. A, cells were fixed in methanol for 5 min at 20 °C and labeled with rabbit AMAP2 polyclonal antibodies coupled with a Cy2-conjugated anti-rabbit IgG antibody (a), or live cells expressing EGFP-AMAP2 at a low level were analyzed by detecting the autofluorescence from the tag (b). B, 1 g each of pcDNA-Xpress-amphiphysin IIm and pEGFP-AMAP2 were co-transfected with 0.5 g of pcDNA-Arf6-HA and its mutants (a–p) or pcDNA-Arf1-HA and its mutants (q–x), as indicated. HA-Arfs and Xpress-amphiphysin IIm were detected using an HA polyclonal antibody coupled with a Cy5-conjugated anti-rabbit IgG antibody and an anti-Xpress monoclonal antibody coupled with a Cy3-conjugated anti-mouse IgG antibody, respectively. EGFP-AMAP2 was detected by its autofluorescence. The right panels show merged views of the left three images. C, protein colocalization was quantified as indicated, according to a method described under “Experimental Procedures.” Results were shown as mean values S.E. from three independent experiments. D, cells transfected with 4 g of pEGFP-AMAP2 and 2 g of pcDNA-Arf6Q67L-HA were subjected to immunoelectron microscopy analysis using with anti-HA (a) or anti-GFP antibodies (b). E, cells were transfected with 0.5 g of pcDNA-Arf6Q67L-HA (a–d) or pcDNA-wild type Arf6-HA (e–h), as indicated. Twenty-four h later, cells were fixed with paraformaldehyde. Endogenous AMAP2 and amphiphysin IIm were detected by an anti-AMAP2 rabbit polyclonal antibody coupled with a Cy2-conjugated anti-rabbit IgG antibody or an anti-amphiphysin IIm rat monoclonal antibody coupled with a Cy3-conjugated anti-rat IgM antibody. Arf6-HA proteins were detected using an HA monoclonal antibody coupled with a Cy5-conjugated anti-mouse IgG antibody. The right panels show merged views of the left three images. F, cells were transfected with 0.5 g of pcDNA-Arf6-HA. Twenty-four h after transfection, cells were starved of serum for another 24 h (a–c) and then stimulated with 10 ng/ml epidermal growth factor for 7 min (d–f) before fixation with paraformaldehyde. Endogenous AMAP2 was detected by an AMAP2 rabbit polyclonal antibody coupled with a Cy2-conjugated anti-rabbit IgG antibody. Arf6-HA proteins were detected using an HA monoclonal antibody coupled with a Cy5-conjugated anti-mouse IgG antibody. The right panels show merged views of the left two images. Antibodies that can clearly label endogenous Arf6 in HeLa cells were not available. In B, D, E, and F, images were acquired using laser confocal microscopy by adjusting the focus 3.0 m above the surface of the glass chamber plate, which was near the center of the nucleus of the majority of cells. Bars, 10 m in A, B, E, and F; 200 nm in D.

Article Snippet: Mouse monoclonal anti-Arf6 antibody and rabbit polyclonal anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Labeling, Expressing, Transfection, Immuno-Electron Microscopy, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

doi: 10.1074/jbc.m404196200

Figure Lengend Snippet: FIG. 4. Involvement of AMAP2 in Tac internalization in HeLa cells. A–C, cells were treated with 50 nM of siRNA oligonucleotide duplexes, each designed for Arf6, AMAP2, or APAP2 silencing, as indicated. Twelve h later, the cells were transfected with 0.4 g of the pKCR-Tac plasmid and 0.1 g of pEGFP-C1 and incubated for a further 24 h before being subjected to endocytosis assays using an anti-Tac antibody (B) or biotinylated Tfn (C) as probes. Oligonucleotides with an irrelevant sequence (irr) were included as a negative control. Protein knockdown was assessed by immunoblotting, as indicated (A). siRNA treatment did not affect protein expression levels of Tac (data not shown). D and E, cells were co-transfected with 0.4 g of pKCR-Tac and 1.6 g of plasmids encoding EGFP-AMAP2 (AMAP2), its ArfGAP do- main deletion mutant (ArfGAP), EGFP-APAP2 (APAP2), K44A mu- tant of EGFP-dynamin-2 (Dyn (K44A)), or EGFP alone, as indicated. They were then subjected to endocytosis assays of Tac (D) and Tfn (E). For co-overexpression of wild type Arf6-HA together with EGFP- AMAP2, 0.3 g of pcDNA-Arf6-HA was included (AMAP2 Arf6). Endocytic blockage in transfection-positive cells was defined as de- scribed under “Experimental Procedures.” Results are means S.E. from three independent experiments. *, p 0.01 against values of the control.

Article Snippet: Mouse monoclonal anti-Arf6 antibody and rabbit polyclonal anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Incubation, Sequencing, Negative Control, Knockdown, Western Blot, Expressing, Mutagenesis, Over Expression, Control

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 Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Membrane ABCA1 level decreased in ApoE4 primary astrocytes compared with ApoE3. A, ABCA1 mRNA level in ApoE3 and ApoE4 immortalized astrocytes was assessed by qRT-PCR. B, Total ABCA1 protein in ApoE3 and ApoE4 immortalized astrocytes were detected by Western blot. C, Densitometric quantification total ABCA1 protein of blotting shown in B from three independent experiments. D, ABCA1 mRNA level in ApoE3 and ApoE4 primary astrocytes was assessed by qRT-PCR. E, Membrane (MEM) and total ABCA1 and ApoE protein in ApoE3 and ApoE4 primary astrocytes were detected by Western blot. Na/K+ ATPase was used as the membrane protein loading control. In this immunoblot, the membrane fraction loaded was prepared from 3/8 of total cell volume. The loaded total fraction was 1/10 of total cell lysate volume. Actin was used as the total protein loading control. F, G, Densitometric quantification of membrane (F) and total (G) ABCA1 and ApoE protein of blotting shown in (E) from three independent experiments. H, ARF6 protein in ApoE3 and ApoE4 primary astrocytes was detected by Western blot. I, Densitometric quantification of blotting shown in (H) from three independent experiments. ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Quantitative RT-PCR, Western Blot

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Effect of ApoE isoforms on ABCA1-mediated cholesterol efflux and ApoE lipidation. A, B, ApoE3 or ApoE4 primary astrocyte were seeded overnight in 96-well plates and then incubated with 3H-labeled cholesterol for 24 h. After washing with PBS, DMEM containing 2 mg/ml BSA were added to the wells and cholesterol efflux was measured at 4 h (A) and 24 h (B). Values showed are normalized to the ApoE3 group. C, ApoE4 mouse primary astrocytes were treated with ARF6 siRNA for 48 h and ARF6 expression was measured by Western blot. D, E, ApoE4 primary astrocyte were treated with ARF6 siRNA followed by cholesterol efflux measurement at 4 h (D) and 24 h (E). (F, G) BHK cells were labeled with 3H-cholesterol followed by induction of ABCA1 expression, and then cholesterol efflux was determined after treatment with 0.2 μm recombinant ApoE3 or ApoE4 (F) or with 15 μl of CSF from non-ε4 (n = 9), ε3/ε4 (n = 8), and ε4/ε4 carriers (n = 3) (G) for 4 h. H, ApoE HDL particles in astrocyte conditioned medium (ACM) from ApoE3 or ApoE4 immortalized astrocytes were analyzed by native-PAGE followed by anti ApoE Western blot. ApoE particles were separated into five distinct sizes, very large (α0>1000KDa), large (α1∼720 KDa), medium (α2∼480 KDa), small (α3∼242 KDa) and very small (α4∼35 KDa). I–M, Densitometric quantification of different size of ApoE particles of blotting shown in H from three independent experiments. **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Incubation, Labeling, Expressing, Western Blot, Recombinant, Clear Native PAGE

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Effect of CS-6253 on ABCA1 expression and trafficking. A, ApoE4 primary astrocytes were treated with CS-6253 peptide (1 μm) for 4 h and then the membrane and total cellular protein were collected. ABCA1, ApoE, and ARF6 were detected by Western blot. B, C, Quantification of membrane ABCA1 and ApoE (B) and total ABCA1 and ARF6 (C) by Western blot shown in A from three independent experiments. D, ApoE4 immortalized astrocytes were treated with CHX with and without CS-6253 for 2 and 4 h. ABCA1 protein expression in cell lysates was detected by Western blot. E, Quantification of Western blot shown in D from three independent experiments. ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Effect of CS-6253 on cholesterol efflux function. A, ApoE4 primary astrocytes were incubated with 3H-labeled cholesterol for 24 h. The cells were induced to express ABCA1 by GW3965 compound for 18 h. After washing with PBS, cholesterol efflux were determined after treatment with or without CS-6253 (1 μm) for 4 h. B, ApoE4 mouse primary astrocytes were treated with ARF6 siRNA or scramble siRNA followed by labeling with 3H-cholesterol for 24 h. After washing with PBS, cholesterol efflux was measured after the addition of CS-6253 for 4 h. C, D, BHK cells were labeled with 3H-cholesterol and ABCA1 expression was induced by mifepristone for 18 h. Cholesterol efflux was measured after treatment with 0.2 μm rApoE4 with or without 1 μm CS-6253 (C) or after treatment with 1 μm CS-6253 plus CSF from ε3/ε3 (n = 7), ε3/ε4 (n = 8), or ε4/ε4 (n = 3) carriers (D) for 4 h. **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Incubation, Labeling, Expressing

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Illustration showing the interaction of ApoE isoforms with ABCA1. A, The interaction of ApoE with ABCA1 favors lipidation of ApoE to the form ApoE HDL. B, ApoE4 is prone to forming protein aggregates and activates ARF6 that in turn decreases ABCA1 recycling; lowering ABCA1-mediated lipidation of ApoE. CS-6253 promotes ABCA1 recycling to the plasma membrane and favors ApoE lipidation, counteracting the effects of ApoE4.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques:

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

Journal: Nature Communications

Article Title: CNK2 promotes cancer cell motility by mediating ARF6 activation downstream of AXL signalling

doi: 10.1038/s41467-023-39281-z

Figure Lengend Snippet: a – c Binding of CNK2A with CYTH1, CYTH3 and SAMD12 promotes ARF6 activity. ARF6-GTP was precipitated using GGA3 protein-binding domain (PBD) beads. U2OS cells were depleted of endogenous CNKs, CYTHs or SAMD12 by shRNA and shRNA-insensitive GFP-CNK2A WT or mutant constructs were expressed where indicated. Bar graphs indicate the normalized ratio of GTP-ARF6 to the amount of total ARF6. d Overexpression of FLAG-CYTH1 WT, but not FLAG-CYTH1_E157K, rescued the migration delay observed in CNK2-depleted U2OS cells in wound healing assays. e Quantification of the migration assays shown in ( d ). f Expression of constitutively active (Q67L) or fast cycling (T157A) ARF6-V5, but not the dominant negative mutant (T27N), rescued the migration delay observed in CNK2-depleted U2OS cells in wound healing assays. g Quantification of the migration assays shown in ( f ). h – m GTP-bound RAC1 ( h , i , l ), RHOA ( j , k ), and ARF6 ( m ) were precipitated using PAK-PBD, Rhotekin-RBD or GGA3-PBD beads as indicated. Migrating U2OS cells were depleted of the indicated proteins by shRNA. Bar graphs indicate the normalized ratio of GTP-bound GTPase to the amount of total proteins. All statistical analyses were one-way ANOVA and were performed on data from at least three independent experiments. For b , i , and j each treatment group was compared to the control (shNT). Error bars correspond to mean values ± SD; ns, not significant ( P > 0.05). Scale bar: d , f 200 µm. Source data are provided as a Source Data file.

Article Snippet: Membranes were blocked for at least 30 min in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% Tween-20) containing 5% milk (Carnation) or 2% BSA (Sigma-Aldrich) and then incubated with the following primary antibodies for 2 h at RT or overnight at 4 °C: anti-CNK2 from rabbit (1:500; Cedarlane Laboratories; custom-made antibody), anti-α-Tubulin DM1A from mouse (1:10,000; Millipore Sigma; #T9026), anti-CNKSR3 (CNK3) from mouse (1:1000; Abnova; #H00154043), anti-SAMD12 (A-6) from mouse (1:500; Santa Cruz Biotechnology, Inc.; #sc-377123), anti-FLAG M2 from mouse (1:5000; Sigma-Aldrich; #F1804), anti-GFP from rabbit (1:5000; OriGene; #TP401), anti-Phospho-Myosin Light Chain 2 (S19) from rabbit (1:500; Cell Signaling Technology; #3671), anti-Myosin Light Chain 2 from rabbit (1:1000; Cell Signaling Technology; #3672), anti-HGK (MAP4K4) from rabbit (1:1000; Cell Signaling Technology; #3485), anti-TNIK from rabbit (1:1000; GeneTex; #GTX13141), anti-NF2 from rabbit (1:1000; Sigma-Aldrich; #HPA003097), anti-Cytohesin 1 (2E11) (CYTH1) from mouse (1:500; Invitrogen; #MA1-060), anti-Cytohesin-2 (H-7) (CYTH2) from mouse (1:250; Santa Cruz Biotechnology, Inc.; #sc-374640), anti-Cytohesin 3 (CYTH3) from rabbit (1:1000; Abcam; #ab155691); anti-Scribble (SCRIB) from rabbit (1:1000; Cell Signaling Technology; #4475), anti-Cool1/βPix from rabbit (1:1000; Cell Signaling Technology; #4515), anti-β-Arrestin 2 (C16D9) from rabbit (1:1000; Cell Signaling Technology; #3857), anti-RhoA from mouse (1:1000; Cytoskeleton, Inc.; #ARH04), anti-Rac1 from mouse (1:500; Cytoskeleton, Inc.; #ARC03), anti-Cdc42 from mouse (1:500; Cytoskeleton, Inc.; #ACD03), anti-Arf1 from mouse (1:500; Cytoskeleton, Inc.; #ARF01), anti-Arf6 from mouse (1:500; Cytoskeleton, Inc.; #ARF06), anti-Pan Ras from mouse (1:1000; Cytoskeleton, Inc.; #AESA02), anti-Axl (C89E7) from rabbit (1:1000; Cell Signaling Technology; #8661), anti-phospho-Axl (Tyr702) (D12B2) from rabbit (1:1000; Cell Signaling Technology; #5724), anti-GAS6 (D3A3G) from rabbit (1:1000; Cell Signaling Technology; #67202), anti-Akt (pan) (C67E7) from rabbit (1:2000; Cell Signaling Technology; #4691), anti-phospho-Akt (Ser473) (D9E) from rabbit (1:2000; Cell Signaling Technology; #4060), anti-Vimentin (D21H3) from rabbit (1:10,000; Cell Signaling Technology; #5741), anti-SP1 (D4C3) from rabbit (1:1000; Cell Signaling Technology; #9389), anti-MEK1/2 from rabbit (1:2000; Cell Signaling Technology; #9122), anti-Histone H3 (3H1) from rabbit (1:1000; Cell Signaling Technology; #9717), anti-V5 Tag from mouse (1:5000; Invitrogen; #R960-25), anti-His-probe (H15) from rabbit (1:1000; Santa Cruz Biotechnology, Inc.; #sc-803), and anti-Actin (C4) from mouse (1:5000; Millipore Sigma; #MAB1501).

Techniques: Binding Assay, Activity Assay, Protein Binding, shRNA, Mutagenesis, Construct, Over Expression, Migration, Expressing, Dominant Negative Mutation, Control

Journal: Nature Communications

Article Title: CNK2 promotes cancer cell motility by mediating ARF6 activation downstream of AXL signalling

doi: 10.1038/s41467-023-39281-z

Figure Lengend Snippet: a Fractionation of U2OS cell lysates followed by immunoblotting indicated that endogenous CNK2 is detected in the cytoplasmic and membrane fractions. shRNA-mediated depletion of CNK2 confirmed the specificity of our custom-made anti-CNK2 antibody. Compartment-specific markers were probed to validate the different fractions. These markers included: MEK (cytosolic fraction), RAS (membrane fraction), Vimentin (cytoskeletal fraction), SP1 (nuclear-soluble fraction) and Histone H3 (chromatin-bound fraction). b Endogenous CNK2 colocalized with N-cadherin in migrating U2OS cells. Note that CNK2 is also observed at the leading edge of some migrating cells (arrow). Orange and yellow boxes in the upper left image are magnified below. CNK2 was depleted by shRNA to control for signal specificity. c Fractionation experiments of U2OS cell lysates expressing GFP-CNK2A WT or GFP-CNK2A_W591A indicated that mutation in the PH domain abolished the detection of GFP-CNK2A in the membrane fraction. A schematic representation of CNK2A with the PH domain mutation (W591A) is shown below. d Immunoblots from U2OS cells depleted of CNK2 and expressing shRNA-insensitive GFP-CNK2A WT or GFP-CNK2A_W591A. e Expression of shRNA-insensitive GFP-CNK2A WT but not GFP-CNK2A_W591A rescued the migration delay observed in CNK2-depleted U2OS cells in wound healing-like assays. f Quantification of the migration assays shown in ( e ). g – i Overexpression of GFP-CNK2A WT in migrating U2OS cells increased ARF6-GTP and RAC1-GTP levels and decreased RHOA-GTP levels while overexpression of GFP-CNK2A_W591A or GFP-CNK2B had no effect on GTPase activity. GTP-bound ARF6 ( g ), RAC1 ( h ), and RHOA ( i ) were precipitated using GGA3-PBD or PAK-PBD or Rhotekin-RBD beads, respectively. Bar graphs indicate the normalized ratio of GTP-bound GTPase to the amount of total proteins. j ARF6-V5 co-immunoprecipitated with GFP-CNK2A but not with GFP-CNK2A_W591A in HEK293T cells. The ARF6-V5 interaction with GFP-CNK2A was enhanced by co-expression with FLAG-CYTH1. k Fractionation of U2OS cell lysates showing reduced CYTH1 levels in the membrane fraction following CNK2 depletion. l Purified truncated versions of recombinant His-CNK2A, consisting of residues 553-699 (WT or W591A) and containing the PH domain, were used to probe PIP Strips for lipid binding. Bound proteins were detected using anti-His antibodies. m , n Inhibition of PI3K decreased CNK2 localization at the plasma membrane in migrating U2OS cells. Cells were treated with LY294002 (10 µM) for 16 h and stained for endogenous CNK2 and N-cadherin. 100 cells were quantified for each condition. All statistical analyses were performed on data from three independent experiments. One-way ANOVA was used in ( f – i ), and unpaired t -test was used in n . Error bars correspond to mean values ± SD; ns, not significant ( P > 0.05). Scale bar: b , m 20 µm, e 100 µm. Source data are provided as a Source Data file.

Article Snippet: Membranes were blocked for at least 30 min in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% Tween-20) containing 5% milk (Carnation) or 2% BSA (Sigma-Aldrich) and then incubated with the following primary antibodies for 2 h at RT or overnight at 4 °C: anti-CNK2 from rabbit (1:500; Cedarlane Laboratories; custom-made antibody), anti-α-Tubulin DM1A from mouse (1:10,000; Millipore Sigma; #T9026), anti-CNKSR3 (CNK3) from mouse (1:1000; Abnova; #H00154043), anti-SAMD12 (A-6) from mouse (1:500; Santa Cruz Biotechnology, Inc.; #sc-377123), anti-FLAG M2 from mouse (1:5000; Sigma-Aldrich; #F1804), anti-GFP from rabbit (1:5000; OriGene; #TP401), anti-Phospho-Myosin Light Chain 2 (S19) from rabbit (1:500; Cell Signaling Technology; #3671), anti-Myosin Light Chain 2 from rabbit (1:1000; Cell Signaling Technology; #3672), anti-HGK (MAP4K4) from rabbit (1:1000; Cell Signaling Technology; #3485), anti-TNIK from rabbit (1:1000; GeneTex; #GTX13141), anti-NF2 from rabbit (1:1000; Sigma-Aldrich; #HPA003097), anti-Cytohesin 1 (2E11) (CYTH1) from mouse (1:500; Invitrogen; #MA1-060), anti-Cytohesin-2 (H-7) (CYTH2) from mouse (1:250; Santa Cruz Biotechnology, Inc.; #sc-374640), anti-Cytohesin 3 (CYTH3) from rabbit (1:1000; Abcam; #ab155691); anti-Scribble (SCRIB) from rabbit (1:1000; Cell Signaling Technology; #4475), anti-Cool1/βPix from rabbit (1:1000; Cell Signaling Technology; #4515), anti-β-Arrestin 2 (C16D9) from rabbit (1:1000; Cell Signaling Technology; #3857), anti-RhoA from mouse (1:1000; Cytoskeleton, Inc.; #ARH04), anti-Rac1 from mouse (1:500; Cytoskeleton, Inc.; #ARC03), anti-Cdc42 from mouse (1:500; Cytoskeleton, Inc.; #ACD03), anti-Arf1 from mouse (1:500; Cytoskeleton, Inc.; #ARF01), anti-Arf6 from mouse (1:500; Cytoskeleton, Inc.; #ARF06), anti-Pan Ras from mouse (1:1000; Cytoskeleton, Inc.; #AESA02), anti-Axl (C89E7) from rabbit (1:1000; Cell Signaling Technology; #8661), anti-phospho-Axl (Tyr702) (D12B2) from rabbit (1:1000; Cell Signaling Technology; #5724), anti-GAS6 (D3A3G) from rabbit (1:1000; Cell Signaling Technology; #67202), anti-Akt (pan) (C67E7) from rabbit (1:2000; Cell Signaling Technology; #4691), anti-phospho-Akt (Ser473) (D9E) from rabbit (1:2000; Cell Signaling Technology; #4060), anti-Vimentin (D21H3) from rabbit (1:10,000; Cell Signaling Technology; #5741), anti-SP1 (D4C3) from rabbit (1:1000; Cell Signaling Technology; #9389), anti-MEK1/2 from rabbit (1:2000; Cell Signaling Technology; #9122), anti-Histone H3 (3H1) from rabbit (1:1000; Cell Signaling Technology; #9717), anti-V5 Tag from mouse (1:5000; Invitrogen; #R960-25), anti-His-probe (H15) from rabbit (1:1000; Santa Cruz Biotechnology, Inc.; #sc-803), and anti-Actin (C4) from mouse (1:5000; Millipore Sigma; #MAB1501).

Techniques: Fractionation, Western Blot, Membrane, shRNA, Control, Expressing, Mutagenesis, Migration, Over Expression, Activity Assay, Immunoprecipitation, Purification, Recombinant, Binding Assay, Inhibition, Clinical Proteomics, Staining

Journal: Nature Communications

Article Title: CNK2 promotes cancer cell motility by mediating ARF6 activation downstream of AXL signalling

doi: 10.1038/s41467-023-39281-z

Figure Lengend Snippet: a Plasma membrane localization of CNK2 was strongly reduced by serum starvation or inhibition of the AXL receptor tyrosine kinase (RTK). Migrating U2OS cells were either serum starved or treated with 1 µM of the indicated RTK inhibitors. For statistical analysis, each treatment group was compared to the control (DMSO). b – d Depletion of AXL in U2OS cells with two different shRNAs strongly decreased CNK2 localization at the plasma membrane. e – g Stimulation of serum-starved U2OS cells with 200 ng/mL GAS6, for 30 min, increased phosphorylation of AXL (Y702) and AKT (S473) ( e ), and triggered PI3K-dependent recruitment of CNK2 to the plasma membrane ( f , g ). N-cadherin staining was used as a marker of the plasma membrane. Line scans correspond to the average ± SD immunofluorescence signal intensity for CNK2 in the cytoplasm and at the plasma membrane of two adjacent cells ( n = 30 line scans for each condition). Bold lines indicate the average signal, while the thinner lines represent the standard deviation. All signals were normalized to the strongest signal for each channel. h Migration of U2OS cells towards 10% FBS and 200 ng/mL GAS6 in the transwell assay was strongly reduced by CNK2 depletion. i Quantification of the average number of cells per field for the transwell migration assay shown in ( h ). j Depletion of CNK2 suppressed GAS6-dependent activation of ARF6. Migrating U2OS cells were serum starved for 24 hours and stimulated with 200 ng/mL GAS6 for 30 min. Bar graphs indicate the normalized ratio of GTP-ARF6 to total ARF6 or pAKT (S473) to total AKT. k Proposed model. The scaffold protein CNK2 promotes cancer cell motility by mediating ARF6 activation downstream of AXL signalling (see details in the text). All statistical analyses were one-way ANOVA and were performed on data from three independent experiments. In a , d , g , 100 cells were quantified for each condition. Error bars correspond to mean values ± SD; ns, not significant ( P > 0.05). Scale bar: b , f 20 µm, h 200 µm. Source data are provided as a Source Data file.

Article Snippet: Membranes were blocked for at least 30 min in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% Tween-20) containing 5% milk (Carnation) or 2% BSA (Sigma-Aldrich) and then incubated with the following primary antibodies for 2 h at RT or overnight at 4 °C: anti-CNK2 from rabbit (1:500; Cedarlane Laboratories; custom-made antibody), anti-α-Tubulin DM1A from mouse (1:10,000; Millipore Sigma; #T9026), anti-CNKSR3 (CNK3) from mouse (1:1000; Abnova; #H00154043), anti-SAMD12 (A-6) from mouse (1:500; Santa Cruz Biotechnology, Inc.; #sc-377123), anti-FLAG M2 from mouse (1:5000; Sigma-Aldrich; #F1804), anti-GFP from rabbit (1:5000; OriGene; #TP401), anti-Phospho-Myosin Light Chain 2 (S19) from rabbit (1:500; Cell Signaling Technology; #3671), anti-Myosin Light Chain 2 from rabbit (1:1000; Cell Signaling Technology; #3672), anti-HGK (MAP4K4) from rabbit (1:1000; Cell Signaling Technology; #3485), anti-TNIK from rabbit (1:1000; GeneTex; #GTX13141), anti-NF2 from rabbit (1:1000; Sigma-Aldrich; #HPA003097), anti-Cytohesin 1 (2E11) (CYTH1) from mouse (1:500; Invitrogen; #MA1-060), anti-Cytohesin-2 (H-7) (CYTH2) from mouse (1:250; Santa Cruz Biotechnology, Inc.; #sc-374640), anti-Cytohesin 3 (CYTH3) from rabbit (1:1000; Abcam; #ab155691); anti-Scribble (SCRIB) from rabbit (1:1000; Cell Signaling Technology; #4475), anti-Cool1/βPix from rabbit (1:1000; Cell Signaling Technology; #4515), anti-β-Arrestin 2 (C16D9) from rabbit (1:1000; Cell Signaling Technology; #3857), anti-RhoA from mouse (1:1000; Cytoskeleton, Inc.; #ARH04), anti-Rac1 from mouse (1:500; Cytoskeleton, Inc.; #ARC03), anti-Cdc42 from mouse (1:500; Cytoskeleton, Inc.; #ACD03), anti-Arf1 from mouse (1:500; Cytoskeleton, Inc.; #ARF01), anti-Arf6 from mouse (1:500; Cytoskeleton, Inc.; #ARF06), anti-Pan Ras from mouse (1:1000; Cytoskeleton, Inc.; #AESA02), anti-Axl (C89E7) from rabbit (1:1000; Cell Signaling Technology; #8661), anti-phospho-Axl (Tyr702) (D12B2) from rabbit (1:1000; Cell Signaling Technology; #5724), anti-GAS6 (D3A3G) from rabbit (1:1000; Cell Signaling Technology; #67202), anti-Akt (pan) (C67E7) from rabbit (1:2000; Cell Signaling Technology; #4691), anti-phospho-Akt (Ser473) (D9E) from rabbit (1:2000; Cell Signaling Technology; #4060), anti-Vimentin (D21H3) from rabbit (1:10,000; Cell Signaling Technology; #5741), anti-SP1 (D4C3) from rabbit (1:1000; Cell Signaling Technology; #9389), anti-MEK1/2 from rabbit (1:2000; Cell Signaling Technology; #9122), anti-Histone H3 (3H1) from rabbit (1:1000; Cell Signaling Technology; #9717), anti-V5 Tag from mouse (1:5000; Invitrogen; #R960-25), anti-His-probe (H15) from rabbit (1:1000; Santa Cruz Biotechnology, Inc.; #sc-803), and anti-Actin (C4) from mouse (1:5000; Millipore Sigma; #MAB1501).

Techniques: Clinical Proteomics, Membrane, Inhibition, Control, Phospho-proteomics, Staining, Marker, Immunofluorescence, Standard Deviation, Migration, Transwell Assay, Transwell Migration Assay, Activation Assay