anti gefh1  (Hycult Biotech)

Journal: Journal of Cell Science

Article Title: Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells

doi: 10.1242/jcs.258557

Figure Lengend Snippet: GEF-H1 and ZO-1 colocalisation after stimulation is cingulin dependent. (A) Cingulin-overexpressing (CGN-GFP) and control cells (GFP) were stained for ZO-1 (grey) and GEF-H1 (red) after stimulation with histamine (10 μg/ml) for 15 min. Nuclei were stained with DAPI (blue). Arrows highlight junctional localisation of GEF-H1. Scale bars: 10 μm. (B) The colocalisation of ZO-1 and GEF-H1 was quantified by determining junctional correlation coefficients. (C) Top, CGN-GFP (green) cells were stained for GEF-H1 (red) after 15 min of histamine (10 μg/ml) stimulation. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Immunofluorescence intensity was quantified along the white arrow. Middle, magnified view of indicated area in the top images. Scale bars: 2 μm. Bottom, immunofluorescence intensity graphs are shown for cingulin (green) and GEF-H1 (red) for the position denoted by the arrow. (D) Corresponding junctional correlation coefficients indicate the colocalisation of cingulin and GEF-H1 in CGN-GFP cells after 15 min stimulation with histamine (10 μg/ml), VEGF-A (50 ng/ml) or thrombin (0.5 U/ml). Data are presented as the mean±s.d. * P <0.05, ** P <0.01, *** P ≤0.001 (one-way ANOVA with Tukey's post-hoc test).

Article Snippet: The following primary antibodies were used: anti-cingulin (HPA027586 at 1:2000 dilution for immunofluorescence and HPA027657 at 1:500 for western blot; Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin (A2228, 1:5000; Sigma-Aldrich), anti-ZO-1 (610966, 1:100; BD Biosciences, San Jose, CA, USA), anti-ppMLC2 (Thr18/Ser19) (3674, 1:1000), anti-MLC2 (3672, 1:1000), anti-pAMPKα (Thr172) (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-phospho-AMPK Substrate Motif [LXRXX(pS/pT)] (5759, 1:1000; all from Cell Signaling Technology, Danvers, MA, USA), anti-GEF-H1 (HM2152, 1:100; Hycult, Uden, Netherlands), anti-VWF (A0082, 1:500; Dako, Glostrup, Denmark), anti-VE-cadherin (IM1597, clone TEA 1/31, Beckman Coulter Immunotech, Marseille, France) anti-PLVAP (RDI-PRO10705, clone PAL-E, 1:10; Research Diagnostics Inc., Flanders, NJ, USA), Alexa Fluor 488-conjugated anti-claudin-5 (352588, 1:100; Thermo Fischer Scientific, Waltham, MA, USA) and Alexa Fluor 647-conjugated anti-podoplanin (337008, 1:100; Biolegend, San Diego, CA, USA).

Techniques: Staining, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells

doi: 10.1242/jcs.258557

Figure Lengend Snippet: The junctional colocalisation of GEF-H1 and cingulin after stimulation is dependent on S131, S134, and S149. (A) Left, cingulin (green) and GEF-H1 (red) are shown in CGN-overexpressing cells (CGN-GFP) and phospho-dead (CGN mut S>A) and phosphomimetic (CGN mut S>D) cingulin mutant cells after 15 min of histamine (10 μg/ml) stimulation. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. Middle, magnified view of indicated area in the images on the left. Scale bars: 2 μm. Right, Immunofluorescence intensity graphs for cingulin (green) and GEF-H1 (red) are shown for the position denoted by the white arrow. (B) Corresponding junctional correlation coefficients indicate the colocalisation of cingulin and GEF-H1 after 15 min stimulation. Data are presented as the mean±s.d. ( n =7–10). ** P <0.01, *** P ≤0.001 (one-way ANOVA with Tukey's post-hoc test).

Article Snippet: The following primary antibodies were used: anti-cingulin (HPA027586 at 1:2000 dilution for immunofluorescence and HPA027657 at 1:500 for western blot; Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin (A2228, 1:5000; Sigma-Aldrich), anti-ZO-1 (610966, 1:100; BD Biosciences, San Jose, CA, USA), anti-ppMLC2 (Thr18/Ser19) (3674, 1:1000), anti-MLC2 (3672, 1:1000), anti-pAMPKα (Thr172) (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-phospho-AMPK Substrate Motif [LXRXX(pS/pT)] (5759, 1:1000; all from Cell Signaling Technology, Danvers, MA, USA), anti-GEF-H1 (HM2152, 1:100; Hycult, Uden, Netherlands), anti-VWF (A0082, 1:500; Dako, Glostrup, Denmark), anti-VE-cadherin (IM1597, clone TEA 1/31, Beckman Coulter Immunotech, Marseille, France) anti-PLVAP (RDI-PRO10705, clone PAL-E, 1:10; Research Diagnostics Inc., Flanders, NJ, USA), Alexa Fluor 488-conjugated anti-claudin-5 (352588, 1:100; Thermo Fischer Scientific, Waltham, MA, USA) and Alexa Fluor 647-conjugated anti-podoplanin (337008, 1:100; Biolegend, San Diego, CA, USA).

Techniques: Mutagenesis, Staining, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells

doi: 10.1242/jcs.258557

Figure Lengend Snippet: Cingulin and GEF-H1 colocalise in inflamed small blood vessels of human skin samples. (A) Healthy and inflamed vessels of human skin tissues were stained for cingulin (green) and GEF-H1 (red). Nuclei were stained with DAPI (blue). Scale bars: 5 μm. (B) Immunofluorescence intensity graphs are shown for cingulin (green) and GEF-H1 (red) for the position denoted by the white arrow. (C) The colocalisation of cingulin and GEF-H1 was quantified based on the corresponding correlation coefficients. Datapoints for the same patient are denoted by use of the same colour ( n =12). Data are presented as the mean±s.d. *** P ≤0.001 (Student's t -test). (D) Schematic diagram of the proposed mechanism of the protective effect of cingulin (created using BioRender.com). AMPK-mediated phosphorylation of cingulin regulates its colocalisation with GEF-H1. Binding between GEF-H1 and cingulin leads to decreased MLC2 phosphorylation and reduced vascular permeability.

Article Snippet: The following primary antibodies were used: anti-cingulin (HPA027586 at 1:2000 dilution for immunofluorescence and HPA027657 at 1:500 for western blot; Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin (A2228, 1:5000; Sigma-Aldrich), anti-ZO-1 (610966, 1:100; BD Biosciences, San Jose, CA, USA), anti-ppMLC2 (Thr18/Ser19) (3674, 1:1000), anti-MLC2 (3672, 1:1000), anti-pAMPKα (Thr172) (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-phospho-AMPK Substrate Motif [LXRXX(pS/pT)] (5759, 1:1000; all from Cell Signaling Technology, Danvers, MA, USA), anti-GEF-H1 (HM2152, 1:100; Hycult, Uden, Netherlands), anti-VWF (A0082, 1:500; Dako, Glostrup, Denmark), anti-VE-cadherin (IM1597, clone TEA 1/31, Beckman Coulter Immunotech, Marseille, France) anti-PLVAP (RDI-PRO10705, clone PAL-E, 1:10; Research Diagnostics Inc., Flanders, NJ, USA), Alexa Fluor 488-conjugated anti-claudin-5 (352588, 1:100; Thermo Fischer Scientific, Waltham, MA, USA) and Alexa Fluor 647-conjugated anti-podoplanin (337008, 1:100; Biolegend, San Diego, CA, USA).

Techniques: Staining, Immunofluorescence, Binding Assay, Permeability

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: Reduced GEF-H1 expression results in increased Rac1 activity and decreased RhoA activity. A. GEF-H1 expression is reduced by lentiviral delivered shRNA. BMBC (231BR3) cells were stably transduced with an shRNA scrambled control (sc) or 1 of 4 distinct shRNA expressing lentivirus. Whole cell lysates were analyzed for GEF-H1 and b1 Integrin expression by Western blotting. Blots were probed for b-actin as a loading control. B. The modulation of Rac1 and RhoA small GTPase activity. BMBC (231BR3) cells were stably transduced by a shRNA scrambled control (sc) or 1 of 4 distinct shRNA expressing lentivirus. Cells were plated on fibronectin-coated dishes, subsequently treated with HPSE (none, latent, or active) (100 ng/ml for 1 hr. at 37°C). Whole cell lysates were analyzed for Rac1 and RhoA activity by G-LISA assays normalized to total Rac1/RhoA protein content (Western blotting), respectively. *p < 0.05; **p < 0.01. Asterisks refer to p values comparing HPSE treatment of the GEF-H1 shRNA clones to the scrambled control (no treatment). G-LISA assays were performed in triplicate. C. Effects of HPSE on Rac1 and RhoA expression. 231BR3 cells were treated with or without A-HPSE or L-HPSE, and whole cell lysates were analyzed for Rac1 and RhoA expression levels by Western blotting. Blots were probed for β-actin as loading control. Refer to the “Materials and Methods” section for additional details.

Article Snippet: Antibodies and dilutions were: 1:500 VCAM1(BBIG-V1 (4B2), BBA5, R&D Systems, Minneapolis, MN, USA), 1:100 GEF-H1 (Cl B4/7, HM2152, Hycult Biotech, Plymouth Meeting, PA, USA), 1:400 tubulin (EP1332Y, ab52866, Abcam, Cambridge, MA, USA), 1:150 SDC4 (ab24511, Abcam), 1:50 PKCα (#2056, Cell Signaling Technology, Danvers, MA, USA), 1:40 phalloidin (594, Invitrogen, Carlsbad, CA, USA), 2.5ug/ml Hoechst 33258 mounting medium with antifade: ProLong (Invitrogen).

Techniques: Expressing, Activity Assay, shRNA, Stable Transfection, Transduction, Western Blot, Clone Assay

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: The modulation of Rac1 and RhoA GTPases by cell treatment with latent or active heparanase (L-HPSE and A-HPSE, respectively). Parental (231P) or brain metastatic (231BR) breast cancer cells were exposed to recombinant human L- or A-HPSE (100ng/ml for 1 hr. at 37°C), cell lysates were then prepared and analyzed accordingly. A. Rac1 and RhoA activities by G-LISA assays normalized to total Rac1/RhoA protein content (Western blotting), respectively. G-LISA assays were performed in triplicate. Statistical comparisons via Student’s paired t-test were made from 231P to 231BR exposed to identical treatments, and differences were statistically significant; **p < 0.01. B. Western blotting analyses of total Rac1 and RhoA, GEF-H1, and associated signaling molecules following L- and A-HPSE cell treatments. Expression of Rac1 guanine exchange factor Tiam1, known to be expressed in BMBC cells and regulated by c-src and FAK (29), and the adapter Grb2, which is a scaffold protein associated with these tyrosine kinases (30), are shown. Blots were probed for β-actin as a loading control. Refer to the “Materials and Methods” section for additional details.

Article Snippet: Antibodies and dilutions were: 1:500 VCAM1(BBIG-V1 (4B2), BBA5, R&D Systems, Minneapolis, MN, USA), 1:100 GEF-H1 (Cl B4/7, HM2152, Hycult Biotech, Plymouth Meeting, PA, USA), 1:400 tubulin (EP1332Y, ab52866, Abcam, Cambridge, MA, USA), 1:150 SDC4 (ab24511, Abcam), 1:50 PKCα (#2056, Cell Signaling Technology, Danvers, MA, USA), 1:40 phalloidin (594, Invitrogen, Carlsbad, CA, USA), 2.5ug/ml Hoechst 33258 mounting medium with antifade: ProLong (Invitrogen).

Techniques: Recombinant, Western Blot, Expressing

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: 231BR3 cell morphology is distinct from that of 231P and 231BR. A. Brightfield images demonstrating morphologies of three BMBC cell lines. Upper panel: BMBC 231P cells. Middle panel: BMBC 231BR cells. Lower panel: BMBC 231BR3 cells. Inserts show representative images of cells at higher magnification. B. Immunofluorescence staining for actin (red), GEF-H1 (green), and nucleus (blue) of 231P, 231BR (panel image mostly shows GEF-H1 overexpressor cells), and 231BR3 breast cancer cells. Cells were treated with human recombinant heparanase either in its latent or active form (100 ng/ml for 1hr. at 37°C). C. Immunofluorescence staining for tubulin (red) and nucleus (blue). Arrows indicate tips of spindle in 231P, 231BR, and 231BR3 breast cancer cells. Controls refer to no HPSE treatment. Refer to the “Materials and Methods” section for additional details. D. Immunofluorescence staining for GEF-H1 (green, panels), SDC4 (green, inserts), and nucleus (blue) for 231P, 231BR, and 231BR3 breast cancer cells. Cells were treated with human recombinant heparanase either in its latent or active form (100ng/ml for 1 hr. at 37°C). Control refers to no HPSE treatment. E. Immunofluorescence staining for SDC4 (red, panels) PKCα (red, inserts), and nucleus (blue) for 231P, 231BR, and 231BR3 breast cancer cell lines. Cells were treated with human recombinant heparanase either in the latent or active form (100ng/ml for 1 hr. at 37°C). Refer to the “Materials and Methods” section for additional details.

Article Snippet: Antibodies and dilutions were: 1:500 VCAM1(BBIG-V1 (4B2), BBA5, R&D Systems, Minneapolis, MN, USA), 1:100 GEF-H1 (Cl B4/7, HM2152, Hycult Biotech, Plymouth Meeting, PA, USA), 1:400 tubulin (EP1332Y, ab52866, Abcam, Cambridge, MA, USA), 1:150 SDC4 (ab24511, Abcam), 1:50 PKCα (#2056, Cell Signaling Technology, Danvers, MA, USA), 1:40 phalloidin (594, Invitrogen, Carlsbad, CA, USA), 2.5ug/ml Hoechst 33258 mounting medium with antifade: ProLong (Invitrogen).

Techniques: Immunofluorescence, Staining, Recombinant

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: 231BR3 high GFP expressing cells in culture subsequent to FACS selection of the scrambled control and the GEF-H1 shRNA clones demonstrate spindle-shaped morphology in culture while GEF-H1 knockdown clones have reduced invasiveness with a spheroid cell morphology. A. Invaded cells stably transduced with the lentivirus scrambled control expressing GFP. B. Invaded 231BR3 GEF-H1 shRNA (clone 1) cells expressing GFP. C. Invaded 231BR3 GEF-H1 shRNA (clone 4) cells expressing GFP. The presence and morphologies of GFP-expressing cells from the underside of chemoinvasion chambers were visualized. Inserts to figures 5A–C indicate cells at higher magnification with apparent morphological changes. D. The in vitro BBB transmigration assay. Quantification of invaded 231BR3 cells, scrambled control, GEF-H1 shRNA (clone 1), or GEF-H1 shRNA (clone 4). Asterisks refer to p values (*p < 0.05, ** p < 0.01), comparing the GEF-H1 shRNA clones to the scrambled control when statistically compared via Student’s paired t-test. In vitro BBB transmigration assays were performed in triplicate. Images were acquired by microscopy using a 10X objective. Cells were identified by eye and manually counted for multiple quadrants within the chamber fields, and their number statistically quantified. Images shown in A–C, although representative, are not directly related to quadrants (fields) for the quantitative assessment of cell invasion by in vitro BBB transmigration assays. Refer to the “Materials and Methods” section for additional details. E. Model of the BBB transmigration assay studying 231BR3 cells.

Article Snippet: Antibodies and dilutions were: 1:500 VCAM1(BBIG-V1 (4B2), BBA5, R&D Systems, Minneapolis, MN, USA), 1:100 GEF-H1 (Cl B4/7, HM2152, Hycult Biotech, Plymouth Meeting, PA, USA), 1:400 tubulin (EP1332Y, ab52866, Abcam, Cambridge, MA, USA), 1:150 SDC4 (ab24511, Abcam), 1:50 PKCα (#2056, Cell Signaling Technology, Danvers, MA, USA), 1:40 phalloidin (594, Invitrogen, Carlsbad, CA, USA), 2.5ug/ml Hoechst 33258 mounting medium with antifade: ProLong (Invitrogen).

Techniques: Expressing, Selection, shRNA, Clone Assay, Stable Transfection, Transduction, In Vitro, Transmigration Assay, Microscopy

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: BMBC demonstrate differential GEF-H1 and PKCα protein expression and HPSE treatment modulated SDC4 CT association. A. GEF-H1 and PKCa are SDC CT associated proteins in HPSE-treated BMBC cells. Whole cell lysates from BMBC cells treated with L- or A-HPSE were immunoblotted for GEF-H1, PKCa, SDC4 or β1Integrin to determine protein expression levels in different BMBC cell lines. Blots were probed for β-actin as a loading control. Data presented are representative of four reproducible experiments. B. The production of GST fusion proteins. Affinity glutathione-S-transferase pulldowns for SDC1 and SDC4 were performed as previously described (11). Figure indicates eluted lysates (1 mg/lane) run on a 15% SDS-PAGE gel under reducing conditions. C. GST pulldown of GEF-H1 by SDC1/4CT. D. GST pulldowns of PKCa by SDC1/4CT. BMBC cells were treated with or without latent or active (L- or A-HPSE) (100ng/ml for 1 hr. at 37°C), then whole cell lysates were generated, passed over GST-SDC1/4 CT fusion protein affinity columns. The same lysates were loaded onto GST protein affinity columns followed by immunoblotting for GEF-H1 or PKCa as controls. Refer to the “Materials and Methods” section for additional details.

Article Snippet: Antibodies and dilutions were: 1:500 VCAM1(BBIG-V1 (4B2), BBA5, R&D Systems, Minneapolis, MN, USA), 1:100 GEF-H1 (Cl B4/7, HM2152, Hycult Biotech, Plymouth Meeting, PA, USA), 1:400 tubulin (EP1332Y, ab52866, Abcam, Cambridge, MA, USA), 1:150 SDC4 (ab24511, Abcam), 1:50 PKCα (#2056, Cell Signaling Technology, Danvers, MA, USA), 1:40 phalloidin (594, Invitrogen, Carlsbad, CA, USA), 2.5ug/ml Hoechst 33258 mounting medium with antifade: ProLong (Invitrogen).

Techniques: Expressing, SDS Page, Generated, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: A model for the proposed mechanisms involving heparanase – induced, GEF-H1 – mediated, regulation of Rac1 and RhoA activities in the cytoskeletal dynamics; and HPSE activities on SDC4/β1 integrin as members of focal adhesion altering cell adhesion of brain metastatic breast cancer cells.

Article Snippet: Antibodies and dilutions were: 1:500 VCAM1(BBIG-V1 (4B2), BBA5, R&D Systems, Minneapolis, MN, USA), 1:100 GEF-H1 (Cl B4/7, HM2152, Hycult Biotech, Plymouth Meeting, PA, USA), 1:400 tubulin (EP1332Y, ab52866, Abcam, Cambridge, MA, USA), 1:150 SDC4 (ab24511, Abcam), 1:50 PKCα (#2056, Cell Signaling Technology, Danvers, MA, USA), 1:40 phalloidin (594, Invitrogen, Carlsbad, CA, USA), 2.5ug/ml Hoechst 33258 mounting medium with antifade: ProLong (Invitrogen).

Techniques: