primary goat polyclonal antibody against human dkk3  (R&D Systems)

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy

doi: 10.1073/pnas.1917608117

Figure Lengend Snippet: DKK3 cleavage utilization as PD biomarker in the phase 1 study following treatment with anti-HtrA1 Fab15H6.v4.D221. The SAD stage consisted of doses of anti-HtrA1 Fab15H6.v4.D221 ranging from 1 to 20 mg ( n = 15). Levels of cleaved DKK3 were assessed by Western blot in aqueous humor of patients at baseline and at multiple time points following anti-HtrA1 treatment as a biomarker of anti-HtrA1 modulation of HtrA1 protease activity. A plot of percent change from baseline for aqueous humor cleaved DKK3 by study day is shown for each treatment group.

Article Snippet: Primary antibodies used include anti-DKK3 biotinylated polyclonal goat antibody (R&D Systems; BAF1118), anti-RBP3 rabbit polyclonal antibody (Proteintech; 14352-1-AP), anti-HtrA1:7816 19G10 mouse IgG2a monoclonal antibody (Genentech), and human CLU biotinylated goat polyclonal antibody (R&D Systems; BAF2937).

Techniques: Biomarker Discovery, Western Blot, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Dickkopf-3 (Dkk3) is protective against ischemic neuronal death. (A) Nissl staining in 10 μm coronal mouse brain sections collected every 320 μm along the rostro-caudal extension of the ischemic lesion in wild-type and Dkk3 knockout mice 1 day after middle cerebral artery occlusion (MCAO). (B) Infarct size in wild-type and Dkk3 knockout mice 1 day, 3 days or 7 days following MCAO. Values are means + SEM. * p < 0.05 vs. wild-type mice (Student’s t -test; day 1: n = 7–9, p = 0.0073, t (14) = 3.135; day 3: n = 4–5, p = 0.0143, t (7) = 3.24; day 7: n = 4–7, p = 0.0387, t (9) = 2.42). (C) Immunohistochemical analysis of Dkk3 in the cerebral cortex of wild-type mice at 6 h and 12 h following MCAO. Low and high magnification images are shown. (D) Double fluorescence immunostaining of Dkk3 and GFAP in the ischemic cortex of wild-type mice 12 h following MCAO. Arrowheads show Dkk3 expression in astrocytes. Arrows show Dkk3 expression in GFAP-negative cells.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Staining, Knock-Out, Immunohistochemical staining, Fluorescence, Immunostaining, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Protective activity of Dkk3 in cultured astrocytes exposed to oxidative damage or glucose deprivation. (A) Flow cytometry analysis (FACS) of live cells in cultured cortical astrocytes from wild-type and Dkk3 knockout mice (C57BL/6J strain) following 1 h of incubation with H 2 O 2 (250 μM). Values are means + SEM ( n = 3–4 per group). p < 0.05 vs. the respective control cultures not exposed to H 2 O 2 (*), or vs. wild-type cultures exposed to H 2 O 2 ( # two-way ANOVA + Fisher’s least significant difference (LSD); genotype, p < 0.001, F (1,11) = 15.79; treatment, p < 0.001, F (1,11) = 28.24; genotype × treatment, F (1,11) = 23.43). (B) Same as in (A) but after exposure to 4 h of glucose deprivation. Values are means + SEM. ( n = 6–9 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,25) = 26.73; treatment, p < 0.001, F (1,25) = 19.33; genotype × treatment, F (1,25) = 18.26). Dkk3 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (C) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p = 0.003, F (1,12) = 8.18; treatment, p = 0.004, F (1,12) = 6.53; genotype × treatment, F (1,12) = 5.82). VEGF protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (D) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,12) = 38.54; treatment, p = 0.003, F (1,12) = 6.60; genotype × treatment, F (1,12) = 6.90). Phospho-AKT protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (E) . Values are means + SEM ( n = 6 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,20) = 19.40; treatment, p = 0.016, F (1,20) = 3.09; genotype × treatment, F (1,20) = 3.93). Bcl2 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (F) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,12) = 25.46; treatment, p = 0.038, F (1,12) = 1.74; genotype × treatment, F (1,12) = 3.93). VEGF protein levels in the whole brain of wild-type and Dkk3 knockout mice are shown in (G) . Values are means + SEM ( n = 3–4 per group). * p < 0.05 vs. wild-type mice (Student’s t-test ; p = 0.0005, t 5 = 7.98). Dkk3 and VEGF protein levels in the whole brain of CD1 and C57BL/6J mice are shown in (H) . Values are means ± SEM ( n = 3 per group). * p < 0.05 vs. CD1 mice (Student’s t-test ; Dkk3, p = 0.0099, t (4) = 4.62; VEGF, p = 0.0384, t (4) = 3.04). (I) FACS analysis of live cells in cultures of cortical astrocytes from CD1 mice incubated for 12 h with hrDkk3 (10 ng/ml) prior to a 1 h-exposure to H 2 O 2 (100 μM). Values are means + SEM ( n = 3 per group). p < 0.05 vs. the respective control cultures not exposed to H 2 O 2 (* p = 0.0001), or vs. the respective cultures not treated with hrDkk3 ( # p = 0.0013; one-way ANOVA + Fisher’s LSD; F (3,8) = 26.2). VEGF protein levels in lysates of cultured astrocytes from CD1 mice treated with hrDkk3 (10 ng/ml) for 12 h are shown in (J) . Values are means ± SEM ( n = 3 per group). * p < 0.05 vs. controls (Student’s t-test ; p = 0.0119, t (4) = 4.38). Ctrl = untreated controls.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Knock-Out, Incubation, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Dkk3 is protective against excitotoxic neuronal death in mixed cortical cultures. Neuronal death in mixed cortical cultures prepared from different mouse genotypes (all C57BL/6J strain) and challenged with 100 μM N -methyl-D-aspartate (NMDA) is shown in (A) . Values are means + SEM ( n = 4–8 per group). p < 0.05 vs. the respective cultures not treated with NMDA (*), or vs. cultures treated with NMDA from wild-type mice in both neurons astrocytes and neurons ( # two-way ANOVA + Fisher’s LSD; genotype, p = 0.002 for astrocytes knockout/neurons knockout; p = 0.026 for astrocytes knockout/neurons wild-type, F (3,40) = 2.06; treatment, p < 0.001 for astrocytes wild-type/neurons wild-type, astrocytes knockout/neurons wild-type and astrocytes knockout/neurons knockout; p = 0.003 for astrocytes wild-type/neurons knockout, F (1,40) = 77.52; genotype × treatment, F (3,40) = 2.07). Neuronal death in cultures from CD1 mice challenged with 45 μM NMDA followed or not by 20-h exposure to hrDkk3 (10 ng/ml) is shown in (B) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. cultures not treated with NMDA (* p < 0.0001) or vs. cultures challenged with NMDA not exposed to hrDkk3 ( p = 0.0009; one-way ANOVA + Fisher’s LSD; F (3,12) = 83.69). Data obtained in cultures from CD1 mice challenged with 80 μM NMDA followed or not by 20-h exposure to hrDkk3 (10 ng/ml) w/wo ZM3238881 (10 μM) is shown in (C) . Values are means + SEM ( n = 5–8 per group). p < 0.05 vs. cultures not treated with NMDA (* p = 0.0005 for NMDA, p < 0.0001 for NMDA+ hrDkk3 + ZM323881, p = 0.0012 for NMDA+ ZM323881) or vs. cultures challenged with NMDA and not exposed to hrDkk3 ( # p = 0.0045; one-way ANOVA + Fisher’s LSD; F (5,32) = 8.39). Neuroprotection by hrVEGF against NMDA toxicity (here, 150 μM) in mixed cortical cultures from CD1 mice is shown in (D) . Cells were exposed to hrVEGF for 20 h following the NMDA pulse. Values are means + SEM ( n = 4–11 per group). p < 0.05 vs. cultures not treated with NMDA (* p < 0.0001) or vs. cultures challenged with NMDA not exposed to hrVEGF ( # p < 0.0001; one-way ANOVA + Fisher’s LSD; F (5,31) = 22.26).

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Knock-Out

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Schematic representation of the protective feedback Dkk3-mediated in astrocytes and/or neurons exposed to cellular stress (glucose deprivation, oxidative stress or ischemia). Dkk3 leads to VEGF upregulation with ensuing VEGFR2 activation, AKT phosphorylation and upregulation of the antiapoptotic factor Bcl2.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Activation Assay, Phospho-proteomics