Journal: Journal of Biological Chemistry

Article Title: SHIP1, an SH2 Domain Containing Polyinositol-5-phosphatase, Regulates Migration through Two Critical Tyrosine Residues and Forms a Novel Signaling Complex with DOK1 and CRKL

doi: 10.1074/jbc.m006250200

Figure Lengend Snippet: FIG. 1. SHIP1 and DOK1 form a complex in Ba/F3.p210 cells re-expressing SHIP1. A-C, SHIP1 expression was induced by doxycycline treatment of Ba/F3.p210.pTRE-SHIP cells. Lysates of untreated cells (2) or cells treated for 18 h with 1 mg/ml doxycycline (1) were used for precipitations. A, lysates of 20 3 106 Ba/F3.p210.pTRE and Ba/F3.p210.pTRE-SHIP cells were incubated with anti-SHIP1 and anti-DOK1 antibodies and SHIP1, DOK1, or tyrosine-phosphorylated proteins were detected by immunoblotting (I.B.). B, lysates of 20 3 106 Ba/ F3.p210.pTRE-SHIP cells were incubated with 5 mg of GST and GST fusion protein of the SHIP1 SH2 or the DOK1 PTB domain immobilized on glutathione beads. Co-precipitation of ABL, SHIP1, or DOK1 was detected by immunoblotting (I.B.). C, lysates of 20 3 106 Ba/F3.p210.pTRE-SHIP cells were immunoprecipitated with antibodies against SHIP1, DOK1, and BCR/ABL as indicated. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Specific direct binding of GST and a GST fusion protein of the DOK1 PTB domain or the SHIP1 SH2 domain to proteins in the immunoprecipitates was detected in a protein overlay assay. The molecular mass of the proteins is indicated in kDa on the left of each figure. D, Ba/F3.p210.TetON cell were transfected with the empty vector (pTRE), a vector containing full-length SHIP1 (pTRE-SHIP), or SHIP1 containing a Y917F (pTRE-SHIP.Y917F) or a Y1020F (pTRE-SHIP.Y1020F) point mutation. SHIP1 protein was induced 1 day after transfectionly by doxycycline treatment for 24 h. SHIP1 expression was detected by immunoblotting (I.B.) in whole cell lysate (2.5 3 105 cells) or in precipitations using a GST-DOK1 PTB domain fusion protein and lysates of 25 3 106 cells. The molecular mass of the proteins is as indicated in kDa on the left of each figure.

Article Snippet: Mouse monoclonal antibodies against SHIP1 (clone P1C1), DOK1 (Santa Cruz Biotechnology, Santa Cruz, CA), ABL (clone 3F12), and CRKL (clone 3-5, for immunoblotting only) were used for immunoblotting or immunoprecipitation.

Techniques: Expressing, Incubation, Western Blot, Immunoprecipitation, SDS Page, Membrane, Binding Assay, Overlay Assay, Transfection, Plasmid Preparation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: SHIP1, an SH2 Domain Containing Polyinositol-5-phosphatase, Regulates Migration through Two Critical Tyrosine Residues and Forms a Novel Signaling Complex with DOK1 and CRKL

doi: 10.1074/jbc.m006250200

Figure Lengend Snippet: FIG. 3. SHIP1 associates with CRKL and DOK1 in cells stimu- lated transformed by v-ABL and TEL/ABL. A and B, lysates of Ba/F3, Ba/F3.p210, Ba/F3.TEL-ABL and Ba/F3.v-Abl were used to de- tect tyrosine-phosphorylated proteins (p-Tyr) by immunoblotting (I.B.). The blots were stripped and reprobed with antibodies against SHIP1, p85PI3K (PI3K), DOK1, or CRKL. The molecular mass of the proteins is indicated in kDa on the left of each figure. A, total cell lysates of 2.5 3 105 cells were used for immunoblotting. B, SHIP1 was immunoprecipi- tated from cell lysates of 20 3 106 cells. TEL/ABL and v-Abl are indicated by arrows. C, DOK1 and CRKL were immunoprecipitated (IP) from cell lysates of 20 3 106 cells as indicated.

Article Snippet: Mouse monoclonal antibodies against SHIP1 (clone P1C1), DOK1 (Santa Cruz Biotechnology, Santa Cruz, CA), ABL (clone 3F12), and CRKL (clone 3-5, for immunoblotting only) were used for immunoblotting or immunoprecipitation.

Techniques: Transformation Assay, Western Blot, Immunoprecipitation

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: Journal of Cerebral Blood Flow & Metabolism

Article Title: Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

doi: 10.1177/0271678X17725211

Figure Lengend Snippet: The adverse effects of silencing endogenous CD200R1 or Dok1 by CD200R1 siRNA or Dok1 siRNA at 24 h after GMH. (a) Representative western blot bands. (b) Quantitative analysis of CD200 showed that protein level of CD200 increased following the treatment with CD200Fc. (c) Quantitative analysis of CD200R1 showed CD200R1 increased in the treatment group and decreased in GMH group. CD200R1 siRNA intervention significantly decreased CD200R1 level. However, CD200R1 level does not change with Dok1 siRNA intervention. (d) Quantitative analysis of Dok1 showed that Dok1 expression significantly decreased by both Dok1 siRNA and CD200R1 siRNA intervention. Data are expressed as mean ± SD, *P < 0.05 vs Sham, #P < 0.05 vs vehicle, &P < 0.05 vs Treatment, n = 6/group, one-way ANOVA followed by the Tukey test.

Article Snippet: CD200R1 small interfering RNA (siRNA, 500 pmol, MyBioSource), scrambled siRNA (500 pmol, MyBioSource), and Dok1 siRNA (500 pmol, MyBioSource) in 0.5 μl PBS was infused at 1.0 μl/min through the Hamilton syringe at 24 h prior to GMH induction.

Techniques: Western Blot, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

doi: 10.1177/0271678X17725211

Figure Lengend Snippet: Effects of CD200R1 siRNA and Dok1 siRNA on IL-1beta and TNF-alpha expression in the presence of CD200Fc (1.5 mg/kg) at 24 h after GMH. (a) Representative western blots bands. (b) Quantitative analysis of IL-1beta. GMH enhanced while CD200Fc reduced the protein level of IL-1beta. (c) Quantitative analysis of TNF-alpha. GMH enhanced protein level of TNF-alpha while CD200Fc attenuated this induction. Data are expressed as mean ± SD, *P < 0.05 vs Sham, #P < 0.05 vs GMH + Vehicle, &P < 0.05 vs Treatment, n = 6/group, one-way ANOVA followed by the Tukey test.

Article Snippet: CD200R1 small interfering RNA (siRNA, 500 pmol, MyBioSource), scrambled siRNA (500 pmol, MyBioSource), and Dok1 siRNA (500 pmol, MyBioSource) in 0.5 μl PBS was infused at 1.0 μl/min through the Hamilton syringe at 24 h prior to GMH induction.

Techniques: Expressing, Western Blot

Journal: Oncoimmunology

Article Title: Docking protein-1 promotes inflammatory macrophage signaling in gastric cancer

doi: 10.1080/2162402X.2019.1649961

Figure Lengend Snippet:

Article Snippet: Full-length (FL) DOK1 cDNA (aa 1–481; 62 kDa) was inserted into pTarget (Promega, Madison, WI).

Techniques: Virus, Suspension