Structured Review

Abcam fascin
<t>Rab35</t> is required for IL-17A–induced phosphorylation of PKCα and <t>fascin</t> in ASMCs. ( A ) Mouse WT, Act1 KO ASMC, and AMSC infected with lentivirus containing control (ctrl) or Rab35 shRNA were treated with IL-17A for 0, 15, and 30 min. Cell lysates were subjected to Western blot (WB) with indicated Abs. ( B ) Mouse ASMCs were treated with IL-17A for 0, 15, 30, and 60 min. Lysates were subjected to immunoprecipitation (IP) with anti-Rab35 GTP Ab, after which they were analyzed by WB with indicated Abs. ( C ) WT mouse ASMCs were infected with lentivirus containing ctrl or Rab35 shRNA and treated with IL-17A for 0, 15, and 30 min. Lysates were subjected to IP with anti-fascin Ab, after which they were analyzed by WB with indicated Abs. ( D ) Model: upon IL-17A stimulation (orange dots), Rab35/GDP is recruited to IL-17R/Act1 complex, which, in turn, recruits DennD1C, switching Rab35/GDP (purple) to Rab35/GTP (red). While the complex Act1/Rab35/GTP mediates PKCα activation (p-S657; phosphorylation is depicted as red asterisk), DennD1C brings actin/fascin bundles close to the complex. The activated PKCα then interacts with and phosphorylates fascin on Ser39, resulting in the dissociation of fascin from actin bundles. The release of actin bundles from fascin allows them to interact with myosin assembling into stress fibers, generating contraction force.
Fascin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility"

Article Title: IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1801025

Rab35 is required for IL-17A–induced phosphorylation of PKCα and fascin in ASMCs. ( A ) Mouse WT, Act1 KO ASMC, and AMSC infected with lentivirus containing control (ctrl) or Rab35 shRNA were treated with IL-17A for 0, 15, and 30 min. Cell lysates were subjected to Western blot (WB) with indicated Abs. ( B ) Mouse ASMCs were treated with IL-17A for 0, 15, 30, and 60 min. Lysates were subjected to immunoprecipitation (IP) with anti-Rab35 GTP Ab, after which they were analyzed by WB with indicated Abs. ( C ) WT mouse ASMCs were infected with lentivirus containing ctrl or Rab35 shRNA and treated with IL-17A for 0, 15, and 30 min. Lysates were subjected to IP with anti-fascin Ab, after which they were analyzed by WB with indicated Abs. ( D ) Model: upon IL-17A stimulation (orange dots), Rab35/GDP is recruited to IL-17R/Act1 complex, which, in turn, recruits DennD1C, switching Rab35/GDP (purple) to Rab35/GTP (red). While the complex Act1/Rab35/GTP mediates PKCα activation (p-S657; phosphorylation is depicted as red asterisk), DennD1C brings actin/fascin bundles close to the complex. The activated PKCα then interacts with and phosphorylates fascin on Ser39, resulting in the dissociation of fascin from actin bundles. The release of actin bundles from fascin allows them to interact with myosin assembling into stress fibers, generating contraction force.
Figure Legend Snippet: Rab35 is required for IL-17A–induced phosphorylation of PKCα and fascin in ASMCs. ( A ) Mouse WT, Act1 KO ASMC, and AMSC infected with lentivirus containing control (ctrl) or Rab35 shRNA were treated with IL-17A for 0, 15, and 30 min. Cell lysates were subjected to Western blot (WB) with indicated Abs. ( B ) Mouse ASMCs were treated with IL-17A for 0, 15, 30, and 60 min. Lysates were subjected to immunoprecipitation (IP) with anti-Rab35 GTP Ab, after which they were analyzed by WB with indicated Abs. ( C ) WT mouse ASMCs were infected with lentivirus containing ctrl or Rab35 shRNA and treated with IL-17A for 0, 15, and 30 min. Lysates were subjected to IP with anti-fascin Ab, after which they were analyzed by WB with indicated Abs. ( D ) Model: upon IL-17A stimulation (orange dots), Rab35/GDP is recruited to IL-17R/Act1 complex, which, in turn, recruits DennD1C, switching Rab35/GDP (purple) to Rab35/GTP (red). While the complex Act1/Rab35/GTP mediates PKCα activation (p-S657; phosphorylation is depicted as red asterisk), DennD1C brings actin/fascin bundles close to the complex. The activated PKCα then interacts with and phosphorylates fascin on Ser39, resulting in the dissociation of fascin from actin bundles. The release of actin bundles from fascin allows them to interact with myosin assembling into stress fibers, generating contraction force.

Techniques Used: Infection, shRNA, Western Blot, Immunoprecipitation, Activation Assay

2) Product Images from "Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma"

Article Title: Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

Journal: BMC Cancer

doi: 10.1186/1471-2407-12-32

Fascin overexpressed cells demonstrated increase in cell-ECM adhesion and loss of cell-cell contacts . ( A ) Cell adhesion of fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells to different ECM substrates was measured as described. The data shown is the average from three experiments with the mean and standard deviation. ( B ) Western blot analysis of stable fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) clones with antibodies to α6 integrin, β4 integrin and phosphorylated FAK. β-actin and total FAK were used as internal loading controls respectively. ( C ) Representative images of aggregates formed by of fascin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells in hanging drop assay. ( D ) Histogram showing size of aggregates formed by overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells. Mean and standard deviation of 3 independent experiments is plotted ( p
Figure Legend Snippet: Fascin overexpressed cells demonstrated increase in cell-ECM adhesion and loss of cell-cell contacts . ( A ) Cell adhesion of fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells to different ECM substrates was measured as described. The data shown is the average from three experiments with the mean and standard deviation. ( B ) Western blot analysis of stable fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) clones with antibodies to α6 integrin, β4 integrin and phosphorylated FAK. β-actin and total FAK were used as internal loading controls respectively. ( C ) Representative images of aggregates formed by of fascin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells in hanging drop assay. ( D ) Histogram showing size of aggregates formed by overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells. Mean and standard deviation of 3 independent experiments is plotted ( p

Techniques Used: Plasmid Preparation, Standard Deviation, Western Blot, Clone Assay

Overexpression of fascin leads to reorganization of actin cytoskeleton . ( A ) Western blot and confocal analysis of stable overexpression of Fascin-GFP (AW-Fascin-1 and AW-Fascin-2) and empty vector control pEGFP (AW-GFP-Cont) clones derived from AW13516 cells with antibodies to GFP and fascin. β-actin was used as loading control. Scale bars: 10 μm. ( B ) Representative confocal images of stably expressed GFP tagged fascin and GFP alone in AW-Fascin-1, AW-Fascin-2 and AW-GFP-Cont clones respectively. Cells were counter stained with DAPI. Scale bars: 10 μm. Arrow heads and arrows indicate filopodia and lamellipodia respectively. ( C ) Representative confocal images of F-actin stained with phalloidin-TRITC and the co-localization of F-actin with Fascin-GFP in stable AW-Fascin-1, AW-Fascin-2 and AW-GFP-Cont clones. Cells were counter stained with DAPI. Scale bars: 10 μm. Arrows indicate colocalization of Fascin-GFP with F-actin at filopodia like structures. ( D ) Histogram showing number of filopodia formed by fascin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells. Mean and standard deviation of 3 independent experiments is plotted ( p
Figure Legend Snippet: Overexpression of fascin leads to reorganization of actin cytoskeleton . ( A ) Western blot and confocal analysis of stable overexpression of Fascin-GFP (AW-Fascin-1 and AW-Fascin-2) and empty vector control pEGFP (AW-GFP-Cont) clones derived from AW13516 cells with antibodies to GFP and fascin. β-actin was used as loading control. Scale bars: 10 μm. ( B ) Representative confocal images of stably expressed GFP tagged fascin and GFP alone in AW-Fascin-1, AW-Fascin-2 and AW-GFP-Cont clones respectively. Cells were counter stained with DAPI. Scale bars: 10 μm. Arrow heads and arrows indicate filopodia and lamellipodia respectively. ( C ) Representative confocal images of F-actin stained with phalloidin-TRITC and the co-localization of F-actin with Fascin-GFP in stable AW-Fascin-1, AW-Fascin-2 and AW-GFP-Cont clones. Cells were counter stained with DAPI. Scale bars: 10 μm. Arrows indicate colocalization of Fascin-GFP with F-actin at filopodia like structures. ( D ) Histogram showing number of filopodia formed by fascin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells. Mean and standard deviation of 3 independent experiments is plotted ( p

Techniques Used: Over Expression, Western Blot, Plasmid Preparation, Clone Assay, Derivative Assay, Stable Transfection, Staining, Standard Deviation

3) Product Images from "Biophysical characterization of actin bundles generated by the Chlamydia trachomatis Tarp effector"

Article Title: Biophysical characterization of actin bundles generated by the Chlamydia trachomatis Tarp effector

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2018.04.093

Tarp generates flexible actin bundles. ( A ) The average length of the actin structures were measured from the TIRF microscopy images where 500 nM of rhodamine labelled F-actin was incubated with or without 500 nM of Tarp or the mutant Tarp proteins. ( B ) Persistence lengths ( L p ) of actin filaments alone, and Tarp-, FAB domain-, α-actinin- or fascin-mediated actin bundles were analyzed ([actin] = 500 nM, [Tarp, FAB or fascin] = 500 nM, for α-actinin, [actin] = 5 μM and [α-actinin] = 1 μM). The data represent the average of three experiments. ( C ) Persistence length was measured for the actin bundles generated by co-incubation of F-actin and Tarp in three different molar ratios (2:1, 1:1 and 1:3) ([actin] = 500nM). Mann-Whitney test was performed between 2 non-parametric groups. The data plotted as mean ± SD. * = p
Figure Legend Snippet: Tarp generates flexible actin bundles. ( A ) The average length of the actin structures were measured from the TIRF microscopy images where 500 nM of rhodamine labelled F-actin was incubated with or without 500 nM of Tarp or the mutant Tarp proteins. ( B ) Persistence lengths ( L p ) of actin filaments alone, and Tarp-, FAB domain-, α-actinin- or fascin-mediated actin bundles were analyzed ([actin] = 500 nM, [Tarp, FAB or fascin] = 500 nM, for α-actinin, [actin] = 5 μM and [α-actinin] = 1 μM). The data represent the average of three experiments. ( C ) Persistence length was measured for the actin bundles generated by co-incubation of F-actin and Tarp in three different molar ratios (2:1, 1:1 and 1:3) ([actin] = 500nM). Mann-Whitney test was performed between 2 non-parametric groups. The data plotted as mean ± SD. * = p

Techniques Used: Microscopy, Incubation, Mutagenesis, Generated, MANN-WHITNEY

4) Product Images from "β-Catenin/CBP–Dependent Signaling Regulates TGF-β–Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells"

Article Title: β-Catenin/CBP–Dependent Signaling Regulates TGF-β–Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.16-20162

Schematic representation of the β-catenin–mediated mechanisms that regulate TGF-β–induced EMT in LECs. TGF-β–induced downregulation of E-cadherin leads to disruption of E-cadherin/β-catenin complex that results in interaction of β-catenin with CBP. Free β-catenin may interact with either Smad 67 in a CBP-dependent manner or TCF, and regulate TGF-β–induced α-SMA, fascin, and MMP9 expression. Inhibition of interaction between β-catenin and TCF fails to inhibit TGF-β–induced EMT-like changes in LECs. However, inhibition of interaction between β-catenin and CBP prevents TGF-β–induced EMT in lens explants.
Figure Legend Snippet: Schematic representation of the β-catenin–mediated mechanisms that regulate TGF-β–induced EMT in LECs. TGF-β–induced downregulation of E-cadherin leads to disruption of E-cadherin/β-catenin complex that results in interaction of β-catenin with CBP. Free β-catenin may interact with either Smad 67 in a CBP-dependent manner or TCF, and regulate TGF-β–induced α-SMA, fascin, and MMP9 expression. Inhibition of interaction between β-catenin and TCF fails to inhibit TGF-β–induced EMT-like changes in LECs. However, inhibition of interaction between β-catenin and CBP prevents TGF-β–induced EMT in lens explants.

Techniques Used: Expressing, Inhibition

5) Product Images from "Notch4 inhibition reduces migration and invasion and enhances sensitivity to docetaxel by inhibiting Akt/fascin in pancreatic cancer cells"

Article Title: Notch4 inhibition reduces migration and invasion and enhances sensitivity to docetaxel by inhibiting Akt/fascin in pancreatic cancer cells

Journal: Oncology Letters

doi: 10.3892/ol.2016.5097

Notch4 siRNA regulates the expression of p-Akt, Akt, GSK3, p-GSK3 and fascin in pancreatic cancer cells. The protein expression levels were determined by western blot analysis. Akt, p-Akt and fascin protein expression were markedly decreased in the Notch4 siRNA + docetaxel group compared with that in the Notch4 siRNA and docetaxel groups. GAPDH was used as an internal control to demonstrate equal protein loading. Lane 1, blank control; lane 2, control siRNA; lane 3, Notch4 siRNA; lane 4, docetaxel; lane 5, Notch4 siRNA + docetaxel. GSK3, glycogen synthase kinase 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p, phosphorylated; siRNA, small interfering RNA.
Figure Legend Snippet: Notch4 siRNA regulates the expression of p-Akt, Akt, GSK3, p-GSK3 and fascin in pancreatic cancer cells. The protein expression levels were determined by western blot analysis. Akt, p-Akt and fascin protein expression were markedly decreased in the Notch4 siRNA + docetaxel group compared with that in the Notch4 siRNA and docetaxel groups. GAPDH was used as an internal control to demonstrate equal protein loading. Lane 1, blank control; lane 2, control siRNA; lane 3, Notch4 siRNA; lane 4, docetaxel; lane 5, Notch4 siRNA + docetaxel. GSK3, glycogen synthase kinase 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p, phosphorylated; siRNA, small interfering RNA.

Techniques Used: Expressing, Western Blot, Small Interfering RNA

6) Product Images from "Role of RNA-interference-induced zinc finger protein 139 suppression in gastric cancer cell sensitivity to chemotherapeutic agents"

Article Title: Role of RNA-interference-induced zinc finger protein 139 suppression in gastric cancer cell sensitivity to chemotherapeutic agents

Journal: Oncology Letters

doi: 10.3892/ol.2015.3421

Western blot analysis of the expression of ZNF139, PDXK, ANXA2 and fascin in (A) SGC7901 cells and (B) transplanted tumors. 1, blank control group; 2, small interfering RNA-ZNF139 group; 3, negative control group. ZNF139, zinc finger protein 139; PDXK,
Figure Legend Snippet: Western blot analysis of the expression of ZNF139, PDXK, ANXA2 and fascin in (A) SGC7901 cells and (B) transplanted tumors. 1, blank control group; 2, small interfering RNA-ZNF139 group; 3, negative control group. ZNF139, zinc finger protein 139; PDXK,

Techniques Used: Western Blot, Expressing, Small Interfering RNA, Negative Control

7) Product Images from "WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion"

Article Title: WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion

Journal: Scientific Reports

doi: 10.1038/srep23590

WIP is strongly expressed in invasive basal-B BCC. ( a ) Relationship between WIP mRNA levels and the invasive behavior of breast cancer cells (BCC). Microarray data for WIP gene expression were retrieved from two reports 35 36 and BCC lines were grouped according to their invasive potential, as described 35 . ( b ) MDA-MB-231 and MCF-7 cells were cultured on mouse peritoneal BM (4 d). After fixing in 4% PFA, samples were stained for IF for mouse type IV collagen (red), F-actin (green) and nuclei (DAPI, blue) and visualized by confocal microscopy. Bars: 25 μm. ( c ) Lysates of basal-B (red) and luminal cells (green) were analyzed by WB using anti-WIP, -WIRE, -N-WASP, -cortactin and -fascin antibodies, with GAPDH expression as control (not shown). Protein expression values were normalized to the highest value in each graph. Data show mean ± SD of at least three independent experiments. ns, not significant; *p
Figure Legend Snippet: WIP is strongly expressed in invasive basal-B BCC. ( a ) Relationship between WIP mRNA levels and the invasive behavior of breast cancer cells (BCC). Microarray data for WIP gene expression were retrieved from two reports 35 36 and BCC lines were grouped according to their invasive potential, as described 35 . ( b ) MDA-MB-231 and MCF-7 cells were cultured on mouse peritoneal BM (4 d). After fixing in 4% PFA, samples were stained for IF for mouse type IV collagen (red), F-actin (green) and nuclei (DAPI, blue) and visualized by confocal microscopy. Bars: 25 μm. ( c ) Lysates of basal-B (red) and luminal cells (green) were analyzed by WB using anti-WIP, -WIRE, -N-WASP, -cortactin and -fascin antibodies, with GAPDH expression as control (not shown). Protein expression values were normalized to the highest value in each graph. Data show mean ± SD of at least three independent experiments. ns, not significant; *p

Techniques Used: Microarray, Expressing, Multiple Displacement Amplification, Cell Culture, Staining, Confocal Microscopy, Western Blot

8) Product Images from "Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells"

Article Title: Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198508

Semiquantitive immunoblot analyses. The expression of vimentin (A, E-G), fascin (B, H-J), focal adhesion kinase (FAK) (C, K-M), and phosphorylated focal adhesion kinase (pFAK Y925 ) (D, N-P) was quantified in U-373 MG wild-type cells and clones overexpressing Slit2 or Robo1 and the effect of photon irradiation on their expression was assessed. Proteins were isolated 24h after irradiation. WT, U-373 MG wild-type cells; Ctrl, U-373 MG control cells (transfected with empty vector); Robo1, U-373 MG-Robo1 (stable clone overexpressing Robo1); Slit2, U-373 MG-Slit2 (stable clone overexpressing Slit2).
Figure Legend Snippet: Semiquantitive immunoblot analyses. The expression of vimentin (A, E-G), fascin (B, H-J), focal adhesion kinase (FAK) (C, K-M), and phosphorylated focal adhesion kinase (pFAK Y925 ) (D, N-P) was quantified in U-373 MG wild-type cells and clones overexpressing Slit2 or Robo1 and the effect of photon irradiation on their expression was assessed. Proteins were isolated 24h after irradiation. WT, U-373 MG wild-type cells; Ctrl, U-373 MG control cells (transfected with empty vector); Robo1, U-373 MG-Robo1 (stable clone overexpressing Robo1); Slit2, U-373 MG-Slit2 (stable clone overexpressing Slit2).

Techniques Used: Expressing, Clone Assay, Irradiation, Isolation, Transfection, Plasmid Preparation, Stable Transfection

9) Product Images from "Foxp3+ T-Regulatory Cells in Sj?gren's Syndrome "

Article Title: Foxp3+ T-Regulatory Cells in Sj?gren's Syndrome

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2008.080246

Immunohistochemical stainings of paraffin-embedded MSG biopsies that were obtained from one patient with SS ( A , B ), non-SS sialadenitis (related to infection by HCV, designated as HCV + ) ( C , D ), and a sicca-complaining control (control) with negative biopsy ( E , F ). Foxp3 + cells displaying the typical nuclear staining ( A , C , E ) were solely detected among the infiltrating mononuclear cells and were located at the areas of CD3 + T cells ( B , D , F ). Double stainings revealed that Foxp3 + cells (brown, indicated by arrows ) were also positive for the T-cell-specific CD3 ( G ) and the CD4 ( H ) antigens (red, arrowheads ), but not the B-cell marker CD20 ( I ), the macrophage-specific CD68 molecule ( J ), or fascin ( K ) and S100 ( L ) proteins that characterize follicular and interdigitating DCs, respectively (red-stained cells, designated by arrowheads ). Representative examples are shown. Original magnifications: ×100 ( A–F ); ×400 ( G–L ).
Figure Legend Snippet: Immunohistochemical stainings of paraffin-embedded MSG biopsies that were obtained from one patient with SS ( A , B ), non-SS sialadenitis (related to infection by HCV, designated as HCV + ) ( C , D ), and a sicca-complaining control (control) with negative biopsy ( E , F ). Foxp3 + cells displaying the typical nuclear staining ( A , C , E ) were solely detected among the infiltrating mononuclear cells and were located at the areas of CD3 + T cells ( B , D , F ). Double stainings revealed that Foxp3 + cells (brown, indicated by arrows ) were also positive for the T-cell-specific CD3 ( G ) and the CD4 ( H ) antigens (red, arrowheads ), but not the B-cell marker CD20 ( I ), the macrophage-specific CD68 molecule ( J ), or fascin ( K ) and S100 ( L ) proteins that characterize follicular and interdigitating DCs, respectively (red-stained cells, designated by arrowheads ). Representative examples are shown. Original magnifications: ×100 ( A–F ); ×400 ( G–L ).

Techniques Used: Immunohistochemistry, Infection, Staining, Marker

Related Articles

Incubation:

Article Title: Effects of dynein light chain Tctex-type 3 on the biological behavior of ovarian cancer
Article Snippet: .. After blocking with Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% nonfat dried milk, membranes were washed with TBST and incubated separately with antibodies against DYNLT3 (1:500; Abcam, Biotechnology), Tubulin (1:2,000; CST, Biotechnology), PCNA (1:2,000; CST, Biotechnology), Ezrin (1:1,000; CST, Biotechnology), Fascin (1:1,000; Abcam, Biotechnology), MMP2 (1:1,000; CST, Biotechnology), and MMP9 (1:1,000; CST, Biotechnology) at 4°C overnight. .. The next day, after washing in TBST, membranes were incubated with secondary antibodies (1:5,000 dilution) for 2h at room temperature.

Article Title: Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion
Article Snippet: .. Membranes were incubated with monoclonal antibodies against N-cadherin (1:1000), β-catenin (1:500), p120 catenin (1:1000), and Rab11 (1:1000; BD Transduction Laboratories, Lexington, KY), troponin T (1:500), α-tubulin (1:500), and myosin (1:2000; Sigma-Aldrich), fascin (1:500; Abcam, Cambridge, MA), and M-cadherin (1:200; NanoTools, Munich, Germany) or with polyclonal antibodies against Rab35 (1:800; gift from A. Echard), myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), GFP (1:800; Rockland Immunochemicals, Gilbertsville, PA), and actin (1:1000; Sigma-Aldrich). .. After washing, membranes were incubated with IRDyeR 680 or 800 secondary antibodies (LI-COR Biosciences, Lincoln, NE).

Article Title: Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells
Article Snippet: .. After blocking with a 3% BSA solution, membranes were incubated with primary antibodies reacting with: Robo1 (1:1000; Cat.No. ab7279), fascin (1:3000: Cat.No. ab78487) (abcam, Cambridge, UK), vimentin (1:1000; Cat.No. .. 9782), focal adhesion kinase (FAK; 1:1000; Cat.No.

other:

Article Title: Foxp3+ T-Regulatory Cells in Sj?gren's Syndrome
Article Snippet: Mouse monoclonal antibodies (mAbs) to human Foxp3 (236A/E7) and fascin (SPM133) were purchased from Abcam (Cambridge, UK). mAbs against CD68 (PG-M1), CD20 (L26), and CD4 (OPD4), as well as rabbit polyclonal antibodies to S100 and CD3 were from DAKO (Glostrup, Denmark).

Chloramphenicol Acetyltransferase Assay:

Article Title: Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells
Article Snippet: .. After blocking with a 3% BSA solution, membranes were incubated with primary antibodies reacting with: Robo1 (1:1000; Cat.No. ab7279), fascin (1:3000: Cat.No. ab78487) (abcam, Cambridge, UK), vimentin (1:1000; Cat.No. .. 9782), focal adhesion kinase (FAK; 1:1000; Cat.No.

Blocking Assay:

Article Title: Effects of dynein light chain Tctex-type 3 on the biological behavior of ovarian cancer
Article Snippet: .. After blocking with Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% nonfat dried milk, membranes were washed with TBST and incubated separately with antibodies against DYNLT3 (1:500; Abcam, Biotechnology), Tubulin (1:2,000; CST, Biotechnology), PCNA (1:2,000; CST, Biotechnology), Ezrin (1:1,000; CST, Biotechnology), Fascin (1:1,000; Abcam, Biotechnology), MMP2 (1:1,000; CST, Biotechnology), and MMP9 (1:1,000; CST, Biotechnology) at 4°C overnight. .. The next day, after washing in TBST, membranes were incubated with secondary antibodies (1:5,000 dilution) for 2h at room temperature.

Article Title: Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells
Article Snippet: .. After blocking with a 3% BSA solution, membranes were incubated with primary antibodies reacting with: Robo1 (1:1000; Cat.No. ab7279), fascin (1:3000: Cat.No. ab78487) (abcam, Cambridge, UK), vimentin (1:1000; Cat.No. .. 9782), focal adhesion kinase (FAK; 1:1000; Cat.No.

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    Abcam fascin 1
    Detection of target genes regulated by miR-145 in CRC cell lines. ( A ) Luciferase activity after transfection with the four wild-type 3′-UTR constructs, miR-145, or miR-control. ( B ) miR-145-binding sites in the 3′-UTR region of <t>Fascin-1;luciferase</t> activity after transfection with mutant 3′-UTR constructs in Fascin-1. The no-insert control (NO), wild-type (WT), and mutated-type (MT) constructs are highlighted with the seed region underlined, and base substitutions are highlighted as bold text. ( C ) Luciferase assays using the mutated vectors in which the specific sites targeted by the miR-145 were deleted. MiR-145 expression levels affect Fascin-1 protein ( D ); and mRNA expression ( E ), in CRC cell lines. Error bars (s.d.) were calcu lated from triplicate samples. * P
    Fascin 1, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam fascin silenced hsc 3 cells
    <t>Fascin</t> knockdown efficiency in SCC-15 and <t>HSC-3</t> cells. Cells were transduced with lentivirus expressing shRNA sequences against fascin (shRNA FSCN cells) and control (shRNA Control cells) as outlined in the methods shRNA FSCN cells showed a marked reduction in both mRNA and protein levels when compared with parental cell and shRNA Control cells.
    Fascin Silenced Hsc 3 Cells, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti fascin1
    Overexpression of β-catenin rescues the phenotype of PKCϵ knockout podocytes. A , murine PKCϵ −/− podocytes were transduced with adenoviral vectors expressing either pAd-Dest, PKCϵ, or β-catenin. Immunofluorescence staining of the actin cytoskeleton with phalloidin and against paxillin was performed. B , the cell size of phalloidin-marked podocytes was assessed in ImageJ for determination of the mean area ( A.U , area unit). C , Western blot analysis of P-cadherin expression in murine podocytes from day ( d ) 0 and day 10. D , PKCϵ −/− podocytes were transduced with adenoviral vectors expressing either pAd-Dest, PKCϵ, or β-catenin and then analyzed for P-cadherin expression with Western blot analysis. E , quantification of P-cadherin band intensity using total protein analysis with Coomassie staining of the membrane. F , quantitative RT-PCR of RNA extracted from cells of the same experiment as in A was used to assess <t>fascin1</t> mRNA expression and normalized to HPRT. G , Western blot analysis of the same experiment as in A for fascin1 protein expression. H , quantification of band intensity by total protein analysis with Coomassie staining (*, p
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    93
    Abcam anti fascin
    NF-κB binds to the <t>fascin</t> promoter in response to IL-6 in a STAT3-dependent manner. (A) MKN45 cells were treated with IL-6 for 30 min and ChIP assays were performed with an NF-κB antibody and primers flanking the potential NF-κB binding site. (B) Results from (A) were quantitated by densitometry and expressed as ChIP/input with the untreated sample. (C) MKN45 cells were transfected with STAT3 siRNA or AG490 and cultured for 3 days. Cells were treated with IL-6 for 30 min and ChIP assays were performed with STAT3 or NF-κB antibodies and primers flanking the potential STAT3/NF-κB binding sites. (D) MKN45 cells were transfected with NF-κB <t>p50</t> siRNA and cultured for 4 days. Whole cell extracts were prepared and western blotting was performed with antibodies against NF-κB p50 and GAPDH. (E) MKN45 cells transfected with control or NF-κB p50 siRNA were treated with IL-6 for 30 min. RNA was subjected to quantitative polymerase chain reaction for fascin and GAPDH. Results were standardized to GAPDH and expressed as fold induction with control siRNA. Values represent the mean ± standard deviation. ** P
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    Image Search Results


    Detection of target genes regulated by miR-145 in CRC cell lines. ( A ) Luciferase activity after transfection with the four wild-type 3′-UTR constructs, miR-145, or miR-control. ( B ) miR-145-binding sites in the 3′-UTR region of Fascin-1;luciferase activity after transfection with mutant 3′-UTR constructs in Fascin-1. The no-insert control (NO), wild-type (WT), and mutated-type (MT) constructs are highlighted with the seed region underlined, and base substitutions are highlighted as bold text. ( C ) Luciferase assays using the mutated vectors in which the specific sites targeted by the miR-145 were deleted. MiR-145 expression levels affect Fascin-1 protein ( D ); and mRNA expression ( E ), in CRC cell lines. Error bars (s.d.) were calcu lated from triplicate samples. * P

    Journal: British Journal of Cancer

    Article Title: MicroRNA-145 inhibits tumour growth and metastasis in colorectal cancer by targeting fascin-1

    doi: 10.1038/bjc.2014.122

    Figure Lengend Snippet: Detection of target genes regulated by miR-145 in CRC cell lines. ( A ) Luciferase activity after transfection with the four wild-type 3′-UTR constructs, miR-145, or miR-control. ( B ) miR-145-binding sites in the 3′-UTR region of Fascin-1;luciferase activity after transfection with mutant 3′-UTR constructs in Fascin-1. The no-insert control (NO), wild-type (WT), and mutated-type (MT) constructs are highlighted with the seed region underlined, and base substitutions are highlighted as bold text. ( C ) Luciferase assays using the mutated vectors in which the specific sites targeted by the miR-145 were deleted. MiR-145 expression levels affect Fascin-1 protein ( D ); and mRNA expression ( E ), in CRC cell lines. Error bars (s.d.) were calcu lated from triplicate samples. * P

    Article Snippet: Tissues were incubated with primary antibody mouse to Fascin-1 (1 : 250) (Abcam) at room temperature for 30 min, then stained using the EnVision Detection Kit, peroxidase/DAB, Rabbit/Mouse (Gene Tech, Shanghai, China).

    Techniques: Luciferase, Activity Assay, Transfection, Construct, Binding Assay, Mutagenesis, Expressing

    Functional effects of Fascin-1 expression on SW620 cells. ( A ) Effective suppression of Fascin-1 protein expression by Fascin-1 siRNA and miR-145 mimics, both individually and combined. ( B ) Significant inhibition of cell growth, migration, and invasion of SW620 cells compared with negative controls. (* P

    Journal: British Journal of Cancer

    Article Title: MicroRNA-145 inhibits tumour growth and metastasis in colorectal cancer by targeting fascin-1

    doi: 10.1038/bjc.2014.122

    Figure Lengend Snippet: Functional effects of Fascin-1 expression on SW620 cells. ( A ) Effective suppression of Fascin-1 protein expression by Fascin-1 siRNA and miR-145 mimics, both individually and combined. ( B ) Significant inhibition of cell growth, migration, and invasion of SW620 cells compared with negative controls. (* P

    Article Snippet: Tissues were incubated with primary antibody mouse to Fascin-1 (1 : 250) (Abcam) at room temperature for 30 min, then stained using the EnVision Detection Kit, peroxidase/DAB, Rabbit/Mouse (Gene Tech, Shanghai, China).

    Techniques: Functional Assay, Expressing, Inhibition, Migration

    Antitumour effects of miR-145 overexpression on CRC xenografts. ( A ) Overexpression of miR-145 inhibits CRC growth in vivo , ( n =8; ** P =0.0034). ( B ) Immunohistochemistry was used to examine the expression of Fascin-1 in tumours either with overexpression of miR-145 or with negative control ( × 200 magnification). ( C ) HE staining of lung tissue isolated from nude mice that had been subcutaneously injected with either LV-miR145-SW620 or LV-miRcontrol-SW620 ( × 100 magnification). The metastasis nodules are indicated by arrows. The table gives the incidences of metastasis in mice that had received subcutaneous tail injections of each cell line. Error bars (s.d.) were calculated from triplicate samples.

    Journal: British Journal of Cancer

    Article Title: MicroRNA-145 inhibits tumour growth and metastasis in colorectal cancer by targeting fascin-1

    doi: 10.1038/bjc.2014.122

    Figure Lengend Snippet: Antitumour effects of miR-145 overexpression on CRC xenografts. ( A ) Overexpression of miR-145 inhibits CRC growth in vivo , ( n =8; ** P =0.0034). ( B ) Immunohistochemistry was used to examine the expression of Fascin-1 in tumours either with overexpression of miR-145 or with negative control ( × 200 magnification). ( C ) HE staining of lung tissue isolated from nude mice that had been subcutaneously injected with either LV-miR145-SW620 or LV-miRcontrol-SW620 ( × 100 magnification). The metastasis nodules are indicated by arrows. The table gives the incidences of metastasis in mice that had received subcutaneous tail injections of each cell line. Error bars (s.d.) were calculated from triplicate samples.

    Article Snippet: Tissues were incubated with primary antibody mouse to Fascin-1 (1 : 250) (Abcam) at room temperature for 30 min, then stained using the EnVision Detection Kit, peroxidase/DAB, Rabbit/Mouse (Gene Tech, Shanghai, China).

    Techniques: Over Expression, In Vivo, Immunohistochemistry, Expressing, Negative Control, Staining, Isolation, Mouse Assay, Injection

    (A) Inverse correlation between miR-145 expression and Fascin-1 mRNA levels in CRC cell lines and tissues ( r =−0.607; P

    Journal: British Journal of Cancer

    Article Title: MicroRNA-145 inhibits tumour growth and metastasis in colorectal cancer by targeting fascin-1

    doi: 10.1038/bjc.2014.122

    Figure Lengend Snippet: (A) Inverse correlation between miR-145 expression and Fascin-1 mRNA levels in CRC cell lines and tissues ( r =−0.607; P

    Article Snippet: Tissues were incubated with primary antibody mouse to Fascin-1 (1 : 250) (Abcam) at room temperature for 30 min, then stained using the EnVision Detection Kit, peroxidase/DAB, Rabbit/Mouse (Gene Tech, Shanghai, China).

    Techniques: Expressing

    Fascin knockdown efficiency in SCC-15 and HSC-3 cells. Cells were transduced with lentivirus expressing shRNA sequences against fascin (shRNA FSCN cells) and control (shRNA Control cells) as outlined in the methods shRNA FSCN cells showed a marked reduction in both mRNA and protein levels when compared with parental cell and shRNA Control cells.

    Journal: Oncotarget

    Article Title: Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma

    doi: 10.18632/oncotarget.20360

    Figure Lengend Snippet: Fascin knockdown efficiency in SCC-15 and HSC-3 cells. Cells were transduced with lentivirus expressing shRNA sequences against fascin (shRNA FSCN cells) and control (shRNA Control cells) as outlined in the methods shRNA FSCN cells showed a marked reduction in both mRNA and protein levels when compared with parental cell and shRNA Control cells.

    Article Snippet: Analysis of filopodia-related proteins Paxillin, focal adhesion kinase (FAK) and vinculin expression in fascin-silenced HSC-3 cells and controls was assessed by western blot, using the following antibodies: anti-paxillin (diluted 1:500; Abcam Inc, USA), anti-phospho paxillin (diluted 1:500; Cell Signaling, USA), anti-FAK (diluted 1:500; Millipore, USA), anti-phospho-FAK (diluted 1:500; Cell Signalling, USA), anti-vinculin (diluted 1:500; Sigma-Aldrich, USA), anti-GAPDH (diluted 1:500; Sigma-Aldrich, USA).

    Techniques: Transduction, Expressing, shRNA

    Fascin downregulation inhibits the invasion of SCC-15 and HSC-3 cells in the myoma organotypic invasion model (A) The knockdown of fascin markedly reduced the invasion properties of SCC-15 and HSC-3 cells in the myoma organotypic model when compared with control cells. The invasion depth (B) and the invasion area (C) were significantly reduced for HSC-3 shRNA FSCN cells. *p

    Journal: Oncotarget

    Article Title: Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma

    doi: 10.18632/oncotarget.20360

    Figure Lengend Snippet: Fascin downregulation inhibits the invasion of SCC-15 and HSC-3 cells in the myoma organotypic invasion model (A) The knockdown of fascin markedly reduced the invasion properties of SCC-15 and HSC-3 cells in the myoma organotypic model when compared with control cells. The invasion depth (B) and the invasion area (C) were significantly reduced for HSC-3 shRNA FSCN cells. *p

    Article Snippet: Analysis of filopodia-related proteins Paxillin, focal adhesion kinase (FAK) and vinculin expression in fascin-silenced HSC-3 cells and controls was assessed by western blot, using the following antibodies: anti-paxillin (diluted 1:500; Abcam Inc, USA), anti-phospho paxillin (diluted 1:500; Cell Signaling, USA), anti-FAK (diluted 1:500; Millipore, USA), anti-phospho-FAK (diluted 1:500; Cell Signalling, USA), anti-vinculin (diluted 1:500; Sigma-Aldrich, USA), anti-GAPDH (diluted 1:500; Sigma-Aldrich, USA).

    Techniques: shRNA

    Overexpression of fascin is associated with adhesion, migration, invasion and acquisition of EMT properties (A) Downregulation of fascin significantly induced the adhesive properties of SCC-15 and HSC-3 cells to fibronectin, and of HSC-3 cells on uncoated surfaces. (B) Migration of SCC-15 and HSC-3 cells were significantly decreased by fascin-specific shRNA, as revealed by transwell migration assay. (C) Migration analysis based on scratch wound migration assay showed that fascin-silenced cells (SCC-15 shRNA FSCN and HSC-3 shRNA FSCN) closed the scratch wound significantly more slowly than parental cells (SCC-15 and HSC-3) and shRNA Control cells. (D) Invasion of SCC-15 and HSC-3 cells was significantly inhibited after fascin knockdown. (E) Downregulation of fascin induced significantly the expression of E-cadherin while reducing vimentin expression in SCC-15 cells. For HSC-3 cells, reduction of vimentin expression reached significant levels, while that the induction of E-cadherin did not. *p

    Journal: Oncotarget

    Article Title: Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma

    doi: 10.18632/oncotarget.20360

    Figure Lengend Snippet: Overexpression of fascin is associated with adhesion, migration, invasion and acquisition of EMT properties (A) Downregulation of fascin significantly induced the adhesive properties of SCC-15 and HSC-3 cells to fibronectin, and of HSC-3 cells on uncoated surfaces. (B) Migration of SCC-15 and HSC-3 cells were significantly decreased by fascin-specific shRNA, as revealed by transwell migration assay. (C) Migration analysis based on scratch wound migration assay showed that fascin-silenced cells (SCC-15 shRNA FSCN and HSC-3 shRNA FSCN) closed the scratch wound significantly more slowly than parental cells (SCC-15 and HSC-3) and shRNA Control cells. (D) Invasion of SCC-15 and HSC-3 cells was significantly inhibited after fascin knockdown. (E) Downregulation of fascin induced significantly the expression of E-cadherin while reducing vimentin expression in SCC-15 cells. For HSC-3 cells, reduction of vimentin expression reached significant levels, while that the induction of E-cadherin did not. *p

    Article Snippet: Analysis of filopodia-related proteins Paxillin, focal adhesion kinase (FAK) and vinculin expression in fascin-silenced HSC-3 cells and controls was assessed by western blot, using the following antibodies: anti-paxillin (diluted 1:500; Abcam Inc, USA), anti-phospho paxillin (diluted 1:500; Cell Signaling, USA), anti-FAK (diluted 1:500; Millipore, USA), anti-phospho-FAK (diluted 1:500; Cell Signalling, USA), anti-vinculin (diluted 1:500; Sigma-Aldrich, USA), anti-GAPDH (diluted 1:500; Sigma-Aldrich, USA).

    Techniques: Over Expression, Migration, shRNA, Transwell Migration Assay, Expressing

    Downregulation of fascin does not affect viability or proliferation of SCC-15 and HSC-3 cells Cells were subjected to MTS-cell viability (A and B) and bromodeoxyuridine (BrdU)-labeling cell proliferation (C and D) assays.

    Journal: Oncotarget

    Article Title: Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma

    doi: 10.18632/oncotarget.20360

    Figure Lengend Snippet: Downregulation of fascin does not affect viability or proliferation of SCC-15 and HSC-3 cells Cells were subjected to MTS-cell viability (A and B) and bromodeoxyuridine (BrdU)-labeling cell proliferation (C and D) assays.

    Article Snippet: Analysis of filopodia-related proteins Paxillin, focal adhesion kinase (FAK) and vinculin expression in fascin-silenced HSC-3 cells and controls was assessed by western blot, using the following antibodies: anti-paxillin (diluted 1:500; Abcam Inc, USA), anti-phospho paxillin (diluted 1:500; Cell Signaling, USA), anti-FAK (diluted 1:500; Millipore, USA), anti-phospho-FAK (diluted 1:500; Cell Signalling, USA), anti-vinculin (diluted 1:500; Sigma-Aldrich, USA), anti-GAPDH (diluted 1:500; Sigma-Aldrich, USA).

    Techniques: Labeling

    Overexpression of β-catenin rescues the phenotype of PKCϵ knockout podocytes. A , murine PKCϵ −/− podocytes were transduced with adenoviral vectors expressing either pAd-Dest, PKCϵ, or β-catenin. Immunofluorescence staining of the actin cytoskeleton with phalloidin and against paxillin was performed. B , the cell size of phalloidin-marked podocytes was assessed in ImageJ for determination of the mean area ( A.U , area unit). C , Western blot analysis of P-cadherin expression in murine podocytes from day ( d ) 0 and day 10. D , PKCϵ −/− podocytes were transduced with adenoviral vectors expressing either pAd-Dest, PKCϵ, or β-catenin and then analyzed for P-cadherin expression with Western blot analysis. E , quantification of P-cadherin band intensity using total protein analysis with Coomassie staining of the membrane. F , quantitative RT-PCR of RNA extracted from cells of the same experiment as in A was used to assess fascin1 mRNA expression and normalized to HPRT. G , Western blot analysis of the same experiment as in A for fascin1 protein expression. H , quantification of band intensity by total protein analysis with Coomassie staining (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes

    doi: 10.1074/jbc.M117.775700

    Figure Lengend Snippet: Overexpression of β-catenin rescues the phenotype of PKCϵ knockout podocytes. A , murine PKCϵ −/− podocytes were transduced with adenoviral vectors expressing either pAd-Dest, PKCϵ, or β-catenin. Immunofluorescence staining of the actin cytoskeleton with phalloidin and against paxillin was performed. B , the cell size of phalloidin-marked podocytes was assessed in ImageJ for determination of the mean area ( A.U , area unit). C , Western blot analysis of P-cadherin expression in murine podocytes from day ( d ) 0 and day 10. D , PKCϵ −/− podocytes were transduced with adenoviral vectors expressing either pAd-Dest, PKCϵ, or β-catenin and then analyzed for P-cadherin expression with Western blot analysis. E , quantification of P-cadherin band intensity using total protein analysis with Coomassie staining of the membrane. F , quantitative RT-PCR of RNA extracted from cells of the same experiment as in A was used to assess fascin1 mRNA expression and normalized to HPRT. G , Western blot analysis of the same experiment as in A for fascin1 protein expression. H , quantification of band intensity by total protein analysis with Coomassie staining (*, p

    Article Snippet: Primary antibodies used for Western blot, immunoprecipitation, and immunofluorescent staining were as follows: mouse anti-active-β-catenin (Merck Millipore, Temecula, CA); rabbit anti-β-catenin (Abcam, Cambridge, UK); rabbit anti-phospho-GSK3α/β, rabbit anti-GSK3β, rabbit anti-GFP, and rabbit anti-FLAG (Cell Signaling Technology, Cambridge, UK); rabbit anti-Fascin1 (Abcam, Cambridge, UK), rabbit anti-Paxillin (Merck Millipore); goat anti-P-cadherin (Novus Biologicals, Littleton, CO); and rabbit anti-GST (Cell Signaling Technology).

    Techniques: Over Expression, Knock-Out, Transduction, Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    Schematic overview of β-catenin regulation by PKCϵ in wild-type and PKCϵ −/− podocytes. In wild-type podocytes, PKCϵ regulates cytoplasmic β-catenin level in balance with other PKC isoforms: PKCα phosphorylates β-catenin and, thus, leads to its degradation. PKCϵ inhibits degradation via its interaction sites (Ser-352, Thr-472/Ser-473, and Ser-663) in a GSK3β-independent manner and leads to a predominantly cytosolic localization of β-catenin, which then might bind to the actin-bundling protein fascin1 in filopodia of the podocytes or localize to the membrane, forming the cell adhesion complex with P-cadherin and α-catenin, resulting in a stabilized actin cytoskeleton and cell adhesion. In PKCϵ-deficient podocytes, this balance is disturbed, leading to degradation of β-catenin, abolished P-cadherin expression, and lower fascin1 expression, which then results in impaired cell adhesion and actin bundling and, thus, in a weakened actin cytoskeleton.

    Journal: The Journal of Biological Chemistry

    Article Title: Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes

    doi: 10.1074/jbc.M117.775700

    Figure Lengend Snippet: Schematic overview of β-catenin regulation by PKCϵ in wild-type and PKCϵ −/− podocytes. In wild-type podocytes, PKCϵ regulates cytoplasmic β-catenin level in balance with other PKC isoforms: PKCα phosphorylates β-catenin and, thus, leads to its degradation. PKCϵ inhibits degradation via its interaction sites (Ser-352, Thr-472/Ser-473, and Ser-663) in a GSK3β-independent manner and leads to a predominantly cytosolic localization of β-catenin, which then might bind to the actin-bundling protein fascin1 in filopodia of the podocytes or localize to the membrane, forming the cell adhesion complex with P-cadherin and α-catenin, resulting in a stabilized actin cytoskeleton and cell adhesion. In PKCϵ-deficient podocytes, this balance is disturbed, leading to degradation of β-catenin, abolished P-cadherin expression, and lower fascin1 expression, which then results in impaired cell adhesion and actin bundling and, thus, in a weakened actin cytoskeleton.

    Article Snippet: Primary antibodies used for Western blot, immunoprecipitation, and immunofluorescent staining were as follows: mouse anti-active-β-catenin (Merck Millipore, Temecula, CA); rabbit anti-β-catenin (Abcam, Cambridge, UK); rabbit anti-phospho-GSK3α/β, rabbit anti-GSK3β, rabbit anti-GFP, and rabbit anti-FLAG (Cell Signaling Technology, Cambridge, UK); rabbit anti-Fascin1 (Abcam, Cambridge, UK), rabbit anti-Paxillin (Merck Millipore); goat anti-P-cadherin (Novus Biologicals, Littleton, CO); and rabbit anti-GST (Cell Signaling Technology).

    Techniques: Expressing

    NF-κB binds to the fascin promoter in response to IL-6 in a STAT3-dependent manner. (A) MKN45 cells were treated with IL-6 for 30 min and ChIP assays were performed with an NF-κB antibody and primers flanking the potential NF-κB binding site. (B) Results from (A) were quantitated by densitometry and expressed as ChIP/input with the untreated sample. (C) MKN45 cells were transfected with STAT3 siRNA or AG490 and cultured for 3 days. Cells were treated with IL-6 for 30 min and ChIP assays were performed with STAT3 or NF-κB antibodies and primers flanking the potential STAT3/NF-κB binding sites. (D) MKN45 cells were transfected with NF-κB p50 siRNA and cultured for 4 days. Whole cell extracts were prepared and western blotting was performed with antibodies against NF-κB p50 and GAPDH. (E) MKN45 cells transfected with control or NF-κB p50 siRNA were treated with IL-6 for 30 min. RNA was subjected to quantitative polymerase chain reaction for fascin and GAPDH. Results were standardized to GAPDH and expressed as fold induction with control siRNA. Values represent the mean ± standard deviation. ** P

    Journal: Oncology Letters

    Article Title: Signal transducer and activator of transcription 3 signaling upregulates fascin via nuclear factor-?B in gastric cancer: Implications in cell invasion and migration

    doi: 10.3892/ol.2014.1804

    Figure Lengend Snippet: NF-κB binds to the fascin promoter in response to IL-6 in a STAT3-dependent manner. (A) MKN45 cells were treated with IL-6 for 30 min and ChIP assays were performed with an NF-κB antibody and primers flanking the potential NF-κB binding site. (B) Results from (A) were quantitated by densitometry and expressed as ChIP/input with the untreated sample. (C) MKN45 cells were transfected with STAT3 siRNA or AG490 and cultured for 3 days. Cells were treated with IL-6 for 30 min and ChIP assays were performed with STAT3 or NF-κB antibodies and primers flanking the potential STAT3/NF-κB binding sites. (D) MKN45 cells were transfected with NF-κB p50 siRNA and cultured for 4 days. Whole cell extracts were prepared and western blotting was performed with antibodies against NF-κB p50 and GAPDH. (E) MKN45 cells transfected with control or NF-κB p50 siRNA were treated with IL-6 for 30 min. RNA was subjected to quantitative polymerase chain reaction for fascin and GAPDH. Results were standardized to GAPDH and expressed as fold induction with control siRNA. Values represent the mean ± standard deviation. ** P

    Article Snippet: Antibodies used for western blotting and chromatin immunoprecipitation (ChIP) assays were anti-phosphotyrosine (p)STAT3 (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-STAT3 (Cell Signaling Technology, Inc.), anti-nuclear factor (NF)-κB p50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-fascin (Abcam, Cambridge, UK), anti-hairy and enhancer of split-1 (Hes-1; Abcam), anti-activated-Notch1 (Abcam), anti-activated-Notch2 (Abcam), anti-matrix metalloproteinase (MMP)-2 and anti-MMP-9 (Cell Signaling Technology, Inc.), anti-GAPDH (Abcam), anti-rabbit immunoglobulin (Ig)G and horseradish peroxidase-linked antibody (Cell Signaling Technology, Inc.).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Transfection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation