Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Identification of DOK1 as a potentially hypermethylated gene in EOC cells. A. Semi‐quantitative RT‐PCR validating induction of DOK1 by 5‐aza‐dC treatment in EOC cells. B. BSP DNA methylation analysis of a 308 bp 5′ non‐coding region (nt −1158 to −850) of the DOK1 gene in EOC cells, EOC tumors and normal ovarian tissue samples. Filled circles represent methylated CpGs and open circles represent unmethylated CpGs. CpG plot of the analyzed is also presented (CpGs are indicated with vertical marks).

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Quantitative RT-PCR, DNA Methylation Assay, Methylation

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: DOK1 expression as a function of tissue type (based on signal intensity following IHC).

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Representative IHC images of DOK1 protein expression in A, normal ovarian tissue and B, serous ovarian adenocarcinoma.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Kaplan–Meier curve for progression‐free survival according to the level of DOK1 IHC intensity in tumor samples of 57 serous EOC patients.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques:

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Western blot analysis of DOK1 expression in TOV112 cells. A. DOK1 expression analysis in DOK1‐overexpressing TOV112 clones pCMV‐DOK1(c1–c3), compared to the mock‐transfected clone pCMV. B. DOK1 expression analysis in DOK1 knockdown TOV112 clones shRNA‐DOK1 (sh1‐sh3), compared to the mock‐transduced clone pLKO. β‐actin was used as a loading control.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Western Blot, Expressing, Clone Assay, Transfection, shRNA

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Effect of ectopic DOK1 overexpression or shRNA‐mediated DOK1 knockdown on TOV112 cell migration. Migration was assessed by determining the ability of cells to migrate in a culture plate using a wound‐healing assay after 16–24 h of incubation. A. Effect of DOK1 overexpression on TOV112 cell migration. B. Migration profile of the DOK1‐overexpressing TOV112 clone pCMV‐DOK1(c3), compared with a mock‐transfected TOV112 clone (pCMV‐control). C. Effect of DOK1 knockdown on TOV112 cell migration. D. Migration profile of the DOK1 knockdown TOV112 clone shRNA‐DOK1(sh1), compared with a mock‐transduced TOV112 clone (pLKO‐control). ∗Statistical significance, P < 0.05.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Over Expression, shRNA, Migration, Wound Healing Assay, Incubation, Transfection

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Ectopic alteration of DOK1 expression in TOV112 cells: effect on cell proliferation (colony formation assay). A. Representative images of colony forming assays following DOK1 overexpression. B. Representative images of colony forming assays following DOK1 knockdown. C. Colony formation assay showed a significant increase of colony number in DOK1 stably transfected TOV112 cells, compared to mock‐transfected cells. D. shRNA‐mediated DOK1 knockdown significantly decreased colony number in TOV112 cells, compared to cells, transduced with empty vector. Colonies were counted and colony numbers represent mean of 3 repeats for each clone. Standard deviations are indicated by error bars. ∗Statistical significance, P < 0.05.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing, Colony Assay, Over Expression, Stable Transfection, Transfection, shRNA, Transduction, Plasmid Preparation

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Dose–response cytotoxicity curves upon cisplatin treatment of TOV112 cells following ectopic DOK1 overexpression or shRNA‐mediated DOK1 knockdown. A. Dose–response cytotoxicity curves upon cisplatin treatment of the DOK1‐overexpressing TOV112 clone pCMV‐DOK1(c3). B. Dose–response cytotoxicity curves upon cisplatin treatment of the DOK1 knockdown TOV112 clone shRNA‐DOK1(sh1). Mock transfected/transduced clones and non‐treated TOV112 cells were used as controls. Dose range for cisplatin was 0.05–100 μM. All results were expressed as mean ± SD of three‐independent experiments.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Over Expression, shRNA, Transfection, Clone Assay

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Selected differentially expressed gene groups in TOV112 cells upon DOK1 overexpression or knockdown.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Over Expression, Transduction, shRNA

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Functional analysis for a dataset of differentially expressed genes (≥2‐fold) following DOK1 overexpression and suppression in TOV112 cells. A. Functional analysis of up‐ and down‐regulated genes in TOV21 cells following DOK1 overexpression. B. Functional analysis of up‐ and down‐regulated genes in TOV21 cells following DOK1 suppression. Top functions that meet a p‐value cutoff of 0.05 are displayed.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Functional Assay, Over Expression

Journal: Molecular Oncology

Article Title: Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

doi: 10.1016/j.molonc.2011.07.003

Figure Lengend Snippet: Network analysis of dynamic gene expression in TOV112 cells based on the 2‐fold common gene expression list obtained following ectopic DOK1 overexpression or shRNA‐mediated DOK1 knockdown. A. Network analysis of DOK1‐overexpressing TOV112 cells; B. Network analysis of TOV112 cells following DOK1 suppression. The five top‐scoring networks for each cell line were merged and are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Intensity of the node color indicates the degree of up‐ (red) or downregulation (green). Nodes are displayed using various shapes that represent the functional class of the gene product (square, cytokine, vertical oval, transmembrane receptor, rectangle, nuclear receptor, diamond, enzyme, rhomboid, transporter, hexagon, translation factor, horizontal oval, transcription factor, circle, other). Edges are displayed with various labels that describe the nature of relationship between the nodes: binding only, → acts on. The length of an edge reflects the evidence supporting that node‐to‐node relationship, in that edges supported by article from literature are shorter. Dotted edges represent indirect interaction.

Article Snippet: Ectopic DOK1 expression in TOV112 cells For DOK1 overexpression, the cDNA sequence of the human DOK1 gene (transcript variant 1) cloned in the pCMV6 entry eukaryotic expression vector (pCMV‐DOK1) was purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing, Over Expression, shRNA, Functional Assay, Binding Assay

Journal: bioRxiv

Article Title: Reticular adhesions: A new class of adhesion complex that mediates cell-matrix attachment during mitosis

doi: 10.1101/234237

Figure Lengend Snippet: (A-M) Confocal images of U2OS cells plated for 3 h on vitronectin and immuno-labeled against integrin β5 and: the alpha V (aV) subunit of the αVβ5 heterodimer (A); consensus adhesome components [paxilin (B), FAK (C), zyxin (D), kindlin 2 (E)]; integrin β 5-binding partners [CSK (F), ICAP1 (G), DOK1 (H)]; phospho-tyrosine (I); and cytoskeletal proteins [F-actin (J), cytokeratin (K), beta (β)-tubulin (L), vimentin (M)]. (N) Confocal image of U2OS cells plated for 3 h on laminin (ECM ligand not bound by αVβ5) and immuno-labeled for integrin β5 and vinculin. (O) Confocal images of an unpermeabilised U2OS cell expressing β5-2GFP, immuno-labeled for β5. Scale bars: 10 μm. Boxed areas shown at higher magnification in lower right corners.

Article Snippet: Primary antibodies used for immunofluorescence and/or immuno-blotting include: anti-integrin β5 (15F11; MAB2019Z) (Millipore); anti-integrin αVβ5 (P1F6) (Abcam); polyclonal (rabbit) anti-integrin β5 (ab15459) (Abcam); anti-integrin β5 (4708S) (Cell Signalling Technology); anti-integrin αv (LM142) (Merck Millipore); anti-pan-talin (1 and 2) (53.8) (BioRad); anti-talin 1 (TA205) (Santa Cruz); anti-talin 2 (68E7) (Abcam); anti-integrin αVβ3 (LM609) (Abcam); anti-integrin β3 (AP3) (Abcam); anti-integrin β1 (LM534) (Millipore); anti-vinculin (hVIN-1) (Sigma Aldrich); anti-vinculin (V9131) (Sigma-Aldrich); anti-intersectin 1 (HPA018007) (Atlas Antibodies, Sigma-Aldrich); anti-NUMB (2733) (Cell Signaling Technologies); anti-EPS15L1 (HPA055309) (Atlas Antibodies, Sigma-Aldrich); anti-HIP1 (HPA013606) (Atlas Antibodies, Sigma-Aldrich); anti-WASL (HPA005750) (Atlas Antibodies, Sigma-Aldrich); anti-DAB2 (12906) (Cell Signaling Technologies); anti-paxillin (5H11) (Sigma Aldrich); anti-FAK (BD Biosciences); anti-zyxin (H-200) (Santa Cruz); anti-kindlin 2 (ab74030) (Abcam); anti-ICAP1 (115228) (Abcam); anti-DOK1 (HPA048561) (Atlas Antibodies, Sigma-Aldrich); polyclonal (rabbit) anti-phosphotyrosine (1000) (Cell Signaling); anti-cytokeratin (27988) (Abcam); anti-beta tubulin (DM1A) (Thermo Fisher Scientific); anti-vimentin (8978) (Abcam).

Techniques: Labeling, Binding Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Complement Receptor 3-Mediated Inhibition of Inflammasome Priming by Ras GTPase-Activating Protein During Francisella tularensis Phagocytosis by Human Mononuclear Phagocytes

doi: 10.3389/fimmu.2018.00561

Figure Lengend Snippet: Dok-1 is phosphorylated during infection with serum-opsonized Schu S4 and co-localizes with Ras GTPase-activating protein (RasGAP). Monocyte-derived macrophage monolayers were infected with non-opsonized or pre-opsonized bacteria (MOI of 100). Infection was synchronized at 4°C followed by incubation at 37°C. At the indicated time points, cell lysates were collected and subjected to Western blot for phosphorylated Lyn and Dok-1 (A) . Phosphorylated Dok-1/actin band intensity ratio at different time points from (A) is shown in (D) . Association of RasGAP with phosphorylated Dok-1 was examined by immunoprecipitation using anti-RasGAP Ab (B) . Phosphorylated Dok-1/RasGAP band intensity ratio at different time points from (B) is shown in (D) . Data from (A) and (B) are representative of 3 independent experiments. Co-localization of RasGAP with Dok-1 was examined at the 5 min time point by confocal microscopy (C) . Data are representative photomicrographs from three independent experiments.

Article Snippet: Phospho-Dok-1 (pTyr362) was from Acris Antibodies, Inc. (San Diego, CA, USA).

Techniques: Infection, Derivative Assay, Incubation, Western Blot, Immunoprecipitation, Confocal Microscopy

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) HEK 293 cells were transiently transfected with empty control vector (−) or GFP-Dok1, GFP-Sam68, Myc-BRK or co-transfected with Myc-BRK+GFP-Sam68 and Myc-BRK+GFP-Dok1. Tyrosine phophorylation of cellular proteins were detected in total cell lysates by immunoblot analysis (IB) with anti-phosphotyrosine (anti-pTyr) antibody (PY20). The blots were reprobed with anti-GFP, anti-BRK and anti-β- Tubulin antibodies as a loading control. (B) Tyrosine phosphorylated endogenous Dok1 as confirmed by anti-Dok1 immunoprecipitation (IP) followed by immunoblot analysis with anti-phosphotyrosine antibody and anti-Dok1(top panel). Immunoblot analysis of total cell lysates is showing the expression of Dok1, kinase activity of BRK-WT and BRK-YF, and β-tubulin as a loading control (bottom panel). (C) An in vitro kinase assay was performed using the active kinase, GST-BRK, and the substrate, GST-C-terminus Dok1, in the presence (+) or absence (−) of ATP. Tyrosine phosphorylation was detected using anti-phosphotyrosine antibody. The blots were reprobed with anti-BRK and anti-Dok1 antibody(bottom panel).

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Expressing, Activity Assay, In Vitro, Kinase Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) Schematic diagram of Dok1 showing different deletion and point mutants. (B) The Dok1 deletion mutants and BRK-YF were co-transfected in to HEK 293 cells, the cell were then subjected to immunoprecipitation with anti-GFP antibody followed by immunobloting analysis using anti-phosphotyrosine and anti-GFP antibodies (top panel). Lower panel shows the expression of different GFP-Dok1 deletion mutants, BRK (as input) and β-tubulin as a loading control. (C) Dok1 deletion mutants were transfected either alone or with BRK-YF into HEK 293 cells, the cell lysates were then subjected to immunobloting analysis using antibodies against Dok1, phosphotyrosine, BRK and β-tubulin as loading control. (D) HEK 293 cells were co-transfected with Dok1 point mutants and BRK-YF followed by immunoprecipitation with anti-Dok1 antibody and immunoblotting analysis using anti-phosphotyrosines and anti-Dok1 antibodies. Lower panel shows the expression of BRK, GFP-Dok1 mutants (as input) and β-tubulin as a loading control. (E) HEK 293 cells were cotransfected with BRK-YF and Dok1 point mutants or transfected with BRK-YF alone. Total cell lysates were analyzed by immunoblotting analysis with antibodies against phosphotyrosines, BRK, Dok1 and β-tubulin as loading control.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) HEK 293 cells were transfected with empty vector, Myc-BRK-WT, Myc-BRK-YF, GFP-Dok1 or co-transfected with Myc-BRK-WT/GFP-Dok1 or Myc-BRK-YF/GFP-Dok1 and subjected to immunoprecipitation with anti-Dok1 and immunoblotted with BRK and Dok1 (top 2 panels). The expression of cellular proteins was determined in total cell lysates by immunoblotting for GFP, BRK and β-tubulin as loading control. (B) BRK was immunoprecipitated with anti-BRK and subjected to immunoblotting analysis with anti-phosphotyrosine, anti-Dok1 and anti-BRK antibodies (top panels). Total cell lysates indicate the expression of BRK and Dok1 proteins. (C &D) HEK 293 cells were transfected with GFP-Dok1 alone or cotransfected with the idicated mutants of BRK and subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-BRK and anti-Dok1 antibodies. The cellular proteins were determined from the total cell lysates by immunoblotting analysis with anti-BRK and anti-Dok1 antibodies. (E) Overexpressed GFP-Dok1 or GFP-Dok1-Y362F in HEK 293 cell lysates from GFP-Dok1 or GFP-Dok1-Y362F expressing cells were subjected to pull-down assays with GST alone or recombinant GST-SH3 or GST-SH2 domain of BRK and immunoblotting analysis was performed with anti-Dok1 antibody. (F) GFP-Dok1/BRK-YF or GFP-Dok1-Y362F/BRK-YF cotransfected cohorts of HEK 293 cell lysates were subjected to pull-down assays with GST alone or GST-SH3 or GST-SH2 domain of BRK followed by immunoblotting with anti-Dok1 antibody. (G) Bacterially expressed GST, GST-SH3 and GST-SH2 domain of BRK proteins were detected via Coomassie blue staining.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot, Control, Recombinant, Staining

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-Dok1 and anti-BRK antibodies. β-tubulin expression served as a loading control. (B & C) SKBR3 and BT20 cells were fractionated into the cytosolic, membrane, nuclear and cytoskeleton fractions and subjected to immunoblotting analysis for the detection of BRK and Dok1. β-tubulin and Sam68 were used as controls for the cytosolic/membrane and nuclear compartments, respectively. (D) Stable BRK knockdown was performed on parental breast cancer cell lines SKBR3 using shRNA lentiviral vector plasmids against BRK and analyzed as indicated.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Western Blot, Expressing, Control, Membrane, Knockdown, shRNA, Plasmid Preparation

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) Immunobloting analysis of total cell lysates from HEK-293 stable cell lines is showing the expression of GFP alone' GFP-BRK-WT and GFP-BRK-YF (top panel), BRK (middle panel) and phosphorylated tyrosines (bottom panel). β-tubulin served as a loading control. (B) Immunobloting analysis of endogenous Dok1 in the stable HEK293 sublines. Expression of Dok1 was examined by immunoblotting analysis. (C) Characterization of cell proliferation in response to BRK-WT and BRK-YF. The P -values were determined for control and stably transfected cells and set at *** P ≥0.0001, ** P ≥0.001 and * P ≥0.05 for statistical significance.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Western Blot, Stable Transfection, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A & B) Total RNA was isolated from HEK 293 cells stably transduced with empty vector, GFP, GFP-BRK-WT and GFP-BRK-YF. Levels of Dok1 mRNA were then analyzed using RT-PCR (A) and qPCR (B). RPL13A gene was used as internal control. Error means are ± SEM of three biological repeats each having three technical repeats.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Isolation, Stable Transfection, Transduction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) HEK 293 cells or HEK 293-BRK-YF stable cell line were treated with a protein synthesis inhibitor cyclohexamide (CHX: 200 µg/ml) for the indicated time points and then the cells were lysed and analyzed by immunoblotting for Dok1, BRK and β-tubulin as a loading control. (B) HEK 293 cells were stably transduced with HEK293-BRK-YF and treated with either a proteosome inhibitor MG132 (10 µM) or the vehicle DMSO as the control, at different time points (above the plot). Cellular proteins were determined in total cell lysates by immunoblotting analysis with anti-Dok1, anti-BRK, anti-phosphotyrosine antibodies. β-tubulin was used as a loading control. (C) Empty vector or V-Src was transiently transfected into HEK293 cells and the cells treated with a proteosome inhibitor MG132 (10 µM) and vehicle control DMSO for the indicated time points. Immunoblotting analysis of total cell lysates was performed to detect Dok1, v-Src, phosphotyrosines and β-tubulin served as a loading control. (D & E) HEK 293 cells were transfected with empty control vector or BRK-YF or v-Src and treated with MG132 (10 µM) and Lactacystin (5 µM) or control vehicle for 8 hours. Then the cell lysates were subjected to immunoblot analysis with anti-Dok1 antibody. β-tubulin as a loading control. (F) HEK293-BRK-YF stable cells were transiently cotransfected with Dok1 and HA-Ubiquitin plasmids and after 12 hours the cells were treated MG132 (10 µM) for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-HA and anti-Dok1 antibodies. The inputs were analysed as indicated.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Stable Transfection, Western Blot, Control, Transduction, Plasmid Preparation, Transfection, Ubiquitin Proteomics, Immunoprecipitation

Journal: PLoS ONE

Article Title: BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

doi: 10.1371/journal.pone.0087684

Figure Lengend Snippet: (A) HEK 293 stable sub-cell lines were transduced with mCherry-Dok1 using adenoviral vector. Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-BRK, anti-Dok1, and anti-phosphotyrosine antibodies. β-tubulin served as a loading control. (B & C) HEK 293 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. (D & E) Cell migration determined by the healing of a fixed wound area induced in the different HEK 293 stable transfectant cells. The percentage of open area at 24 h is plotted. (F & G) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: and ** P ≥0.001 and *** P ≥0.0001.

Article Snippet: Recently, Takeda et al. identified Dok1 as a substrate of several tyrosine kinases including BRK .

Techniques: Transduction, Plasmid Preparation, Western Blot, Control, Migration, Transfection, Stable Transfection, Expressing