dkk1 primary antibody  (Proteintech)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 1. In vitro evaluation of the proper effect of the BHQ880 antibody over Dkk1 protein neutralization. (A) Representative images of B1a/7TGC cell reporter line activation when stimulated by the GSK-3 inhibitor CHIR99021 (positive control) or by increasing doses of recombinant mouse Wnt3a (mWnt3a), and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. Non-treated (NT); # vs. 0 ng/mL mWnt3a. (B) Representative images of B1a/7TGC cell reporter line inactivation when previously stimulated cells with 150 ng/mL of mWnt3a were turned off through the addition of increasing doses of recombinant rDkk1, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; # vs. 0 ng/mL rDkk1. (C) Representative images of the BHQ880 neutralizing effect over rDkk1 protein in the B1a/7TGC reporter cells stimulated with 150 ng/mL of mWnt3a, together with 250 ng/mL of rDkk1 and increasing doses of BHQ880 antibody, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; & vs. Wnt3a; # vs 0 ng/mL BHQ880. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between groups. In all cases, scale bars = 50 μm and the data are presented as the mean + SEM. **p <0.01; ***p <0.001; #p <0.05; ##p <0.01; ###p <0.001; &&p <0.01.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: In Vitro, Neutralization, Activation Assay, Positive Control, Recombinant

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 2. Protein expression of Dkk1 in the healthy and injured spinal cord. The figure shows representative images from the immunohistochemical evaluation of Dickkopf-1 (Dkk1) protein expression in the non-lesioned (NL) (n = 5) spinal cord (A) and after SCI at 24 hours post-injury (hpi) (n = 4), and 3 days post-injury (dpi), 7 dpi (n = 4), 14 dpi (n = 5), 28 dpi (n = 3), and 42 dpi (n = 5) at the lesion epicenter (B-G). Dashed line squares in A-G indicate the areas shown in the corresponding higher magnification images. Scale bars = 500 μm. Inserts scale bars = 50 μm. (H) Densitometric analysis of Dkk1 immunolabeling in the NL spinal cord and at the injury epicenter after SCI from 24 hpi to 42 dpi. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between NL and the different times post-injury. In all cases, the data are presented as the mean + SEM. **p <0.01; ***p <0.001.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Expressing, Immunohistochemical staining, Immunolabeling

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 3. Analysis of the circulating levels of Dkk1 in Serum samples from non- lesioned (NL) and lesioned rats at 24 hours after damage (SCI). The figure shows data obtained from the quantitative analysis of serum circulating Dkk1 protein levels measured by ELISA in NL animals (n = 9) and injured animals 24 hours after SCI (n = 9). No significant differences were observed between groups. A two-tailed t test was used to determine the existence of significant differences between groups. The data represent the mean ± SEM.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Neuroscience

Article Title: Minocycline Preserves the Integrity and Permeability of BBB by Altering the Activity of DKK1-Wnt Signaling in ICH Model.

doi: 10.1016/j.neuroscience.2019.06.038

Figure Lengend Snippet: Fig. 4. Mino reduced DKK1 protein expression in rats at 3 days after intracerebral hemorrhage (ICH) by western blot and immunohistochemistry. (A) The expression of DKK1 around the hematoma after intracerebral hemorrhage. Protein expression was normalized to GAPDH and expressed as ratio of sham (n = 3 for each group; *P < 0.05,**P < 0.01). (B) Immunohistochemistry staining showed DKK1 expression around the hematoma after intracerebral hemorrhage (Scale bar represents 100 μm). (C) DKK1 positive cells per high power field according to the immunohistochemistry staining (n = 6 for each group; ***P < 0.01). (D) Immunofluorescence staining showed that DKK1 was co-localized with microglia and neuron, but not co-localized with astrocyte marker (Scale bar represents 20 μm).

Article Snippet: The sections were sequentially incubated at room temperature with the following: 10% normal donkey serum for 30 min, primary antibody rabbit anti-DKK1 antibody (Abcam, catalog: ab109416; 1:300) for 24 h, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, catalog: 111-035-003; 1:5000) for 6 h, and avidin–biotin–peroxidase complex (ABC) (ABC Elite Kit; 1:200; Vector Laboratories) for another 2 h. Peroxidase activity was revealed by dipping the sections for 5 min in a mixture containing 3- diaminobenzidine (DAB) and H2O2 at room temperature.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Marker

Journal: Neuroscience

Article Title: Minocycline Preserves the Integrity and Permeability of BBB by Altering the Activity of DKK1-Wnt Signaling in ICH Model.

doi: 10.1016/j.neuroscience.2019.06.038

Figure Lengend Snippet: Fig. 5. Mino suppresses the expression of DKK1 and the production of inflammatory mediators in micro- glia. 4 × 105 rat primary microglia are plated in 12-well plates and then challenged with LPS for 24 h in the presence and absence of Mino. 200 μl cell-free supernatant was harvested for TNF (A), IL-6 (B) and MMP-9 (C) assay by ELISA. (D) 15 μg total cell lysate was collected after 4 h challenge of LPS and resolved on SDS-PAGT, then immunoblotted with an anti-DKK1 or anti-β-catenin antibody and developed by ECL. Immunoblots were striped Q1 and re-probed with an antibody to GAPDH to ensure equal protein load- ing. “*” and “***” represent significant differences at P < 0.05 and P < 0.001, respectively. Results repre- sent the mean ± SEM of three separate experiments.

Article Snippet: The sections were sequentially incubated at room temperature with the following: 10% normal donkey serum for 30 min, primary antibody rabbit anti-DKK1 antibody (Abcam, catalog: ab109416; 1:300) for 24 h, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, catalog: 111-035-003; 1:5000) for 6 h, and avidin–biotin–peroxidase complex (ABC) (ABC Elite Kit; 1:200; Vector Laboratories) for another 2 h. Peroxidase activity was revealed by dipping the sections for 5 min in a mixture containing 3- diaminobenzidine (DAB) and H2O2 at room temperature.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Neuroscience

Article Title: Minocycline Preserves the Integrity and Permeability of BBB by Altering the Activity of DKK1-Wnt Signaling in ICH Model.

doi: 10.1016/j.neuroscience.2019.06.038

Figure Lengend Snippet: Fig. 8. Schematic model of the mechanism of Mino functions on ICH. Administration of Mino decreases the expression of DKK1 in perihema- tomal tissue or microglia, which enhances the activity of Wnt1, and con- sequently leads to the accumulation of β-catenin. Increased β-catenin results in the increase of Occludin and decreases of inflammatory med- iators including TNF, IL-6 and MMP-9, and ultimately improves the BBB integrity and alleviates the outcomes of ICH.

Article Snippet: The sections were sequentially incubated at room temperature with the following: 10% normal donkey serum for 30 min, primary antibody rabbit anti-DKK1 antibody (Abcam, catalog: ab109416; 1:300) for 24 h, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, catalog: 111-035-003; 1:5000) for 6 h, and avidin–biotin–peroxidase complex (ABC) (ABC Elite Kit; 1:200; Vector Laboratories) for another 2 h. Peroxidase activity was revealed by dipping the sections for 5 min in a mixture containing 3- diaminobenzidine (DAB) and H2O2 at room temperature.

Techniques: Expressing, Activity Assay

Journal: Journal of cell science

Article Title: Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/β-catenin signaling pathway.

doi: 10.1242/jcs.228478

Figure Lengend Snippet: Fig. 1 Dkk1 expression was upregulated in doxorubicin (Dox) treatment in vitro.

Article Snippet: Primary antibodies against Dkk1 (diluted 1:3000,goat) was purchased from R&D systems.

Techniques: Expressing, In Vitro

Journal: Journal of cell science

Article Title: Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/β-catenin signaling pathway.

doi: 10.1242/jcs.228478

Figure Lengend Snippet: Fig. 2 The Changes of Dkk1 in doxorubicin (Dox)-induced cardiotoxicity in vivo.

Article Snippet: Primary antibodies against Dkk1 (diluted 1:3000,goat) was purchased from R&D systems.

Techniques: In Vivo

Journal: Journal of cell science

Article Title: Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/β-catenin signaling pathway.

doi: 10.1242/jcs.228478

Figure Lengend Snippet: Fig. 3 Dkk1 is involved in doxorubicin (Dox) cardiomyopathy in H9C2 cells. H9C2

Article Snippet: Primary antibodies against Dkk1 (diluted 1:3000,goat) was purchased from R&D systems.

Techniques:

Journal: Journal of cell science

Article Title: Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/β-catenin signaling pathway.

doi: 10.1242/jcs.228478

Figure Lengend Snippet: Fig. 4 Overexpression of Dkk1 promoted heart injury induced by doxorubicin

Article Snippet: Primary antibodies against Dkk1 (diluted 1:3000,goat) was purchased from R&D systems.

Techniques: Over Expression

Journal: Journal of cell science

Article Title: Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/β-catenin signaling pathway.

doi: 10.1242/jcs.228478

Figure Lengend Snippet: Fig. 5 Overexpression of Dkk1 exacerbated doxorubicin (Dox)-induced

Article Snippet: Primary antibodies against Dkk1 (diluted 1:3000,goat) was purchased from R&D systems.

Techniques: Over Expression

Journal: Journal of cell science

Article Title: Dkk1 exacerbates doxorubicin-induced cardiotoxicity by inhibiting the Wnt/β-catenin signaling pathway.

doi: 10.1242/jcs.228478

Figure Lengend Snippet: Fig. 7 Dkk1 aggravated doxorubicin (Dox)-induced cardiotoxicity via inhibiting

Article Snippet: Primary antibodies against Dkk1 (diluted 1:3000,goat) was purchased from R&D systems.

Techniques:

Journal: Journal of Cell Communication and Signaling

Article Title: Aberrant Wnt Signaling Pathway in the Hematopoietic Stem/Progenitor Compartment in Experimental Leukemic Animal

doi: 10.1007/s12079-018-0470-6

Figure Lengend Snippet: Deregulation of Wnt signaling pathway in the leukemic hematopoietic stem/progenitor (HSPC) compartment. a-b Representative histogram overlay plots and bar diagrams showed expression levels of canonical (Wnt3a, Fzd7, β-catenin, CyclinD1 and Dkk1) and non-canonical (Wnt5a, Fzd5, Ca2+, CaMKII and ROR2) Wnt signaling pathway components in the control and leukemic HSPC compartment. MFI (Mean Fluorescence Intensity) values indicated significant up-regulation of Wnt3a, Fzd7, β-catenin, CyclinD1 whereas down-regulation of Dkk1, Wnt5a, Fzd5, CaMKII, ROR2 expression and Ca2+ level in the leukemic condition [*P < 0.05; *** < 0.0001]

Article Snippet: Next, the membrane was stained with anti-Dkk1 primary antibody (1:300 dilution; Santa Cruz Biotechnology) followed by HRP tagged secondary antibody (1:600 dilution) and the reaction product was developed using HRP- DAB based chromogenic method.

Techniques: Expressing, Control, Fluorescence

Journal: Journal of Cell Communication and Signaling

Article Title: Aberrant Wnt Signaling Pathway in the Hematopoietic Stem/Progenitor Compartment in Experimental Leukemic Animal

doi: 10.1007/s12079-018-0470-6

Figure Lengend Snippet: Malignant transformation alters BM hematopoietic stromal microenvironment and stroma derived Wnt. a Representative images of dayswise LT-BMC of control and leukemic mice recorded at different time points, viz day 7, 10, 15 and 20. Left panel (inside black border): control BM derived stromal cell generation and their gradual proliferation pattern. Right panel (inside red border): Leukemic BM derived stromal cell generation and their proliferation pattern. Early generation and rapid transformation of stromal precursors to stromal fibroblasts and their higher proliferation potential in leukemic condition [orange arrow: stromal fibroblasts; black arrow: stromal precursors]. b-c Representative ICC images. Wnt3a expression in Bi. Control and Bii. Leukemic BM derived stromal cells. Leukemic stromal cells showed higher Wnt3a protein expression. Wnt5a expression in Ci. Control and Cii. Leukemic BM derived stromal cells. Leukemic stromal cells showed lower Wnt5a protein expression. The brown depositions were the reaction product of substrate DAB and HRP tagged with secondary antibody and its intensity was directly proportional to the protein expression level [Magnification 1000X]. d. Comparative dot blot assay showed decreased level of Dkk1 in the leukemic stromal cell culture supernatant. [red arrow: control; blue arrow: leukemia]

Article Snippet: Next, the membrane was stained with anti-Dkk1 primary antibody (1:300 dilution; Santa Cruz Biotechnology) followed by HRP tagged secondary antibody (1:600 dilution) and the reaction product was developed using HRP- DAB based chromogenic method.

Techniques: Transformation Assay, Derivative Assay, Control, Expressing, Dot Blot, Cell Culture