goat anti human dkk 3  (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