goat anti human dkk 3  (R&D Systems)

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Immunoreactivity of DKK3 and survival curves for patients with invasive serous ovarian cancer. ( a ) DKK3 immunoreactivity in normal epithelium and serous tumors. ( b ) Immunoreactivity of each score in invasive serous carcinoma. ( c ) Kaplan–Meier curves for disease-free survival. Magnification 200×.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques:

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Expression of DKK3 in different ovarian tissue samples.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Expressing

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Clinicopathological parameters and disease-free survival analysis of prognostic factors in 42 patients with serous adenocarcinoma.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Expressing

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Clinicopathological characteristics of women with/without DKK3 expression.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Expressing

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Characteristics of paclitaxel-resistant cells. ( a , b ) Cells were seeded in triplicates in 96-well plates in 0.1 mL culture medium at a density of 1 × 10 4 cells/well. After 24 h, the cells were treated with paclitaxel. MTT assay performed 48 h after treatment showed that the paclitaxel-resistant cells (TOV-21G/PTX and OV-90/PTX) had higher IC 50 than the parental cells. ( c , d ) Paclitaxel treatment reduced the viability of parental cells, while the paclitaxel-resistant cells continued to grow. ( e ) Western blot analysis revealed that DKK3 was lost and that β-catenin and P-glycoprotein were upregulated in paclitaxel-resistant cells (Res) compared to that in their parent cells (Con). β-Actin was used as the loading control. * p < 0.01.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: MTT Assay, Western Blot, Control

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Anti-proliferative effect of the secreted DKK3 on paclitaxel-resistant cells. ( a ) Western blotting results show DKK3 protein levels in the control and DKK3 conditioned medium (CM). MTT assay performed after 24 h of incubation with CM (*** p < 0.001). ( b ) The viability of TOV-21G and TOV-21G/PTX cells after treatment with DKK3 CM diluted to 0%, 50%, and 90%, respectively, with control CM (** p < 0.01). ( c ) The OV-90 and OV-90/PTX cells were incubated with CM with or without DKK3. Western blotting results show DKK3 protein levels in the control and DKK3 CM. MTT assay after 24 h of treatment (*** p < 0.001). ( d ) Western blotting reveals recombinant human DKK3 levels. Two concentrations of the protein (10 μg/mL and 50 μg/mL) were used to treat cells for 72 h (* p < 0.05). ( e , f ) TOV-21G/PTX and OV-90/PTX cells were treated with CM with or without paclitaxel (100 ng/mL and 200 ng/mL, respectively) (** p < 0.01). ( g ) 3D spheroids of OV-90 and OV-90/PTX cells were generated in a microwell array and the images were captured after incubation for 6 d (magnification, 40×; scale bar 300 μm). ( h ) Spheroid viabilities and diameters were compared. The asterisks on the graph denote statistically significant differences ( p < 0.01) between the DKK3 CM group and the control CM group. The diameter of spheroids in the DKK3 CM group was compared to that in the DKK3 CM with paclitaxel group (#, p < 0.01). ( i , j ) OV-90/PTX and TOV-21G/PTX cells were treated as mentioned above and migration rates were evaluated using the migration assay. ( k , l ) The paclitaxel-resistant cells were incubated with control and DKK3 CMs. At the indicated time points, the cells were harvested and subjected to Western blotting. The graphs were plotted based on the band densities measured using the ImageJ software. * p < 0.05.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Western Blot, Control, MTT Assay, Incubation, Recombinant, Generated, Migration, Software

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Secreted DKK3 enhanced paclitaxel susceptibility via inhibition of the β-catenin-P-glycoprotein signaling pathway. ( a , b ) The cells were incubated with control and DKK3 CM. Immunofluorescence analysis after 24 h showed that FLAG-DKK3 was present in the perinuclear area, attenuating TR-non-phospho-β-catenin signaling. Scale bar, 50 μm. ( c , d ) The cells were incubated with control and DKK3 CM and subjected to Western blotting. The graphs were plotted based on the band densities (* p < 0.01). ( e , f ) To activate endogenous β-catenin, cells were treated with LiCl for 24 h and subjected to Western blot analysis. β-Actin was used as the loading control. The intensities of the Western blot bands were normalized to that of β-actin for comparison (* p < 0.01).

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Inhibition, Incubation, Control, Immunofluorescence, Western Blot, Comparison

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: Dkk3 overexpression suppressed the aggressive phenotypes of colorectal cancer cells. After transfection of pcDNA3.1 -Dkk3 , Dkk3 expression became strong in HCT-15 and HCT-116 cells using anti-Dkk3 and anti-His antibodies by Western blot (A) , real-time RT-PCR (B) , and immunofluorescence ( C , green: positive for Dkk3, blue: DAPI for nuclei). The cell culture supernatant of Dkk3 transfectants showed a higher Dkk3 concentration in comparison with those of the control and mock (D) . Colorectal cancer patients showed a higher serum Dkk3 level than healthy individuals regardless of their age, evidenced by ELISA assay (E) . The transfectants showed a lower proliferation (F) and G 2 arrest (G) in comparison with the control and mock. There was an apoptosis-induced effect of Dkk3 overexpression in the transfectants of HCT-15 and HCT-116, evidenced by annexin V assay (H) . JC-1 staining displayed that the mitochondrial membrane potential was decreased in the abovementioned cells with Dkk3 overexpression by fluorescence (I) and flow cytometry (J) . Dkk3-overexpressing cells had a weaker ability to migrate (K) and invade (L) . We also treated the Dkk3 transfectants with anti-Dkk3 antibody (0, 30, and 60 ng/ml) and found that this antibody blocked the effects of Dkk3 overexpression on these phenotypes ( F–L , p < 0.05). mock, cells transfected with pcDNA3.1 vector; * P < 0.05, compared with the control and treatment with anti-Dkk3 antibody.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Cell Culture, Concentration Assay, Comparison, Control, Enzyme-linked Immunosorbent Assay, Annexin V Assay, Staining, Membrane, Fluorescence, Flow Cytometry, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: Recombinant Dkk3 inhibited the aggressive phenotypes of colorectal cancer cells. Treatment with recombinant Dkk3 protein significantly suppressed the proliferation of HCT-15 and HCT-116, compared with the control cells (A) . Recombinant Dkk3 protein could induce G 2 arrest (B) and cell apoptosis (C) of both kinds of cells in a dose-dependent manner. JC-1 staining also demonstrated that recombinant Dkk3 exposure significantly reduced mitochondrial membrane potential of the abovementioned cells treated with 150 ng/mL recombinant protein by fluorescence (D) and flow cytometry (E) . After being exposed to recombinant Dkk3, both colorectal carcinoma cells exhibited a low ability to migrate (F) and invade (G) , compared with the control cells. * P < 0.05, compared with the control.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Recombinant, Control, Staining, Membrane, Fluorescence, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The phenotype-related proteins were screened by Western blot. Dkk3 overexpression and treatment decreased the expression of Bcl-2, cdc25B, cdc25c, N-cadherin, Slug, and Twist and increased the expression of Bax and E-cadherin in HCT-15 and HCT-116 cells. The anti-Dkk3 antibody suppressed the effects of Dkk3 overexpression on the expression of the phenotype-related proteins.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Western Blot, Over Expression, Expressing

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The effects of Dkk3 on tumor growth and carcinogenesis of colorectal cancer. PCR primers were designed (A) and subjected to PCR of tail and stomach DNA (B) with the same color for the corresponding primers and products. Expression of Dkk3, N-cadherin, and E-cadherin was confirmed by real-time PCR (C) and Western blot (D) . Immunohistochemistry was employed to observe the maker proteins’ expression in the intestinal adenoma of target villin-cre/Dkk3 knockout mice induced by sodium desoxycholate (E) . * p < 0.05, compared with the transfectant or knockout mice. WT, wild-type mice; VD, villin-cre +; Dkk3 -/-.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Knock-Out, Transfection

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The clinicopathological significance of Dkk3 mRNA expression in colorectal cancer. Real-time RT-PCR was employed to detect Dkk3 mRNA expression in colorectal cancer samples (A) . Its expression was compared with pathological behaviors of colorectal cancer (B) . N, normal; C, cancer, TA, tubular adenocarcinoma; MA, mucinous adenocarcinoma. * p < 0.05.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The bioinformatics analysis of Dkk3 mRNA expression in colorectal cancer. Oncomine (A) and TCGA (B) data sets were employed to analyze Dkk3 expression in colorectal cancer, and its expression was compared with pathological parameters of cancers. Dkk3 mRNA expression was higher in cancer-associated fibroblasts (CAF) than CD133-positive (CD133+) and -negative (CD133-) colorectal cancer cells (C) . Kaplan-Meier curves were used to analyze the prognostic significance of Dkk3 mRNA expression according to KM plotters (D) . HR, hazard ratio; N, lymph node metastasis. * p < 0.05; **** p < 0.001.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: The clinicopathological significance of Dkk3 protein expression in colorectal cancer. Western blot was employed to detect Dkk3 protein expression in colorectal cancer samples (A) . Its expression was compared with pathological behaviors of colorectal cancer (B) . N, normal; C, cancer, TA, tubular adenocarcinoma; MA, mucinous adenocarcinoma. * p < 0.05.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer

doi: 10.3389/fonc.2020.600322

Figure Lengend Snippet: Dkk3 -enriched signal pathway in colorectal cancer according KEGG analysis.

Article Snippet: After washing with PBS, cells were incubated overnight at 4°C with goat anti-Dkk3 antibody (R&D system) and subsequently with antigoat IgG-FITC antibody (Santa Cruz) at room temperature and then stained with DAPI for nuclear labeling.

Techniques: Migration, Coagulation, Infection