recombinant human wnt5b rwnt5b protein  (R&D Systems)

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: WNT5B is upregulated in the striatal astrocytes of HD patients and HD model mice. ( a ) Heatmap showing the expression levels of WNT signaling-related genes in normal subjects (Normal) and HD patients (HD). Data were extracted from GSE64810 . (b) Expression levels of WNT5B in the prefrontal cortex of 20 HD patients and 49 neuropathologically normal subjects. The error bars represent the means ± SEMs. Student’s t test was used (**, p < 0.01). (c) Western blot analysis of WNT5B protein levels in postmortem striatal tissues from normal and HD patients. (d) Densitometry analysis of WNT5B protein levels ( n = 5 patie n ts per group: normal subjects and HD patients). Student’s t test was performed (***, p < 0.001). (e) Double chromogenic immunostaining images for WNT5B (blue) and GFAP (red) in the striatum of normal and HD patients. Scale bars (black): top, 2 mm; bottom, 10 μm. (f) Quantification of WNT5B immunoreactivity in GFAP-positive cells in the caudate. A total of 15 cells per group were analyzed (5 cells per case, N = 3 cases per group: normal subjects and HD patients). Significantly different at ***, p < 0.001. (g) Quantification of WNT5B immunoreactivity in GFAP-positive cells in the putamen. A total of 15 cells per group were analyzed (5 cells per case, N = 3 cases per group). Significant differences at **, p < 0.01. (h) Volcano plot showing differentially expressed genes in R6/2 HD transgenic mice ( n = 2) compared with WT mice ( n = 2). (i) Heatmap showing the expression levels of WNT signaling-related genes in WT and R6/2 transgenic mice. (j) Immunostaining of WNT5B and S100β in WT and N171-82Q mice. Nuclei were counterstained with DAPI. Scale bars (white): 10 μm. (k) Quantitative analysis of WNT5B immunoreactivity in S100β-positive cells. A total of 15 cells per group were analyzed (5 cells per mouse; N = 3 mice per group: WT and N171-82Q). Significant differences at **, p < 0.01. (l) Experimental design for Western blot analysis of human astrocytes transduced with wild-type HTT (wt HTT ) (25Q) or mutant HTT (m HTT ) (103Q) for 72 h. (m) Western blot analysis of WNT5B protein levels in human astrocytes transfected with wt HTT (25Q) or m HTT (103Q). (n) Densitometry analysis of WNT5B protein levels (3 samples per group: 25Q and 103Q). Significantly different at *, p < 0.05

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Expressing, Western Blot, Immunostaining, Transgenic Assay, Transduction, Mutagenesis, Transfection

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: WNT5B overexpression alters ECM-related gene signatures in HD transgenic mice. ( a ) Experimental design showing the delivery of AAVs (pAAV-GFAP(pro)-Control-mCherry or pAAV-GFAP(pro)-WNT5B-P2A-mCherry) into the dorsal striatum of N171-82Q mice via bilateral stereotaxic injection at 12 weeks of age, followed by RNA-seq analysis of the striatal tissues from three groups of mice (WT, N171-82Q, and N171-82Q + WNT5B). (b) Principal component analysis (PCA) revealed distinct clustering of RNA-seq data among the three groups of mice ( N = 3 mice per group). (c) Volcano plot showing differentially expressed genes in N171-82Q and N171-82Q + WNT5B mice. (d) Venn diagram illustrating the overlap of downregulated genes in N171-82Q mice whose expression was further decreased by WNT5B overexpression. (e) Gene ontology (biological process and cellular component) and KEGG analyses of the 632 genes downregulated in both N171-82Q and N171-82Q + WNT5B mice. (f) Heatmap showing alterations in the expression of cell type-specific ECM-related genes in WT, N171-82Q, and N171-82Q + WNT5B mice ( n = 3 mice per group). (g) Ingenuity pathway analysis (IPA) revealed a molecular network linking WNT signaling with ECM-associated genes in N171-82Q and N171-82Q + WNT5B mice

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Over Expression, Transgenic Assay, Control, Injection, RNA Sequencing, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: MMP14 levels are elevated in HD postmortem brains, and its expression is transcriptionally activated by NFATc2 through the noncanonical WNT signaling pathway in vitro. ( a ) Western blot analysis showing MMP14 protein levels in postmortem striatal tissues from normal subjects and HD patients. ( b ) Densitometry of the MMP14 protein levels ( n = 5 samples per group). Student’s t test (*, p < 0.05). ( c ) Chromogenic staining for MMP14 (blue) and GFAP (brown) in postmortem striatal sections from normal subjects and HD patients. Scale bars: 20 μm. Whole-brain illustrations were created with BioRender.com. ( d ) Quantification of MMP14 immunoreactivity in GFAP-positive astrocytes in the putamen. (36 cells per group; 12 cells per case, N = 3 cases per group). Significantly different at *, p < 0.05. ( e ) Experimental design for the transfection of WNT5B into human astrocytes under normal (HTT-25Q) or HD (mHTT-103Q) conditions for 24 h, followed by mRNA expression analysis via qPCR. ( f ) WNT5B overexpression increased NFATc2 and MMP14 mRNA levels under both normal and HD conditions, whereas CTNNB1 and NFATc4 levels were unchanged. Significantly different at *, p < 0.05. ( g ) Schematic illustration of NFATc2-enriched binding sites (red) in the human MMP14 promoter identified via the TRANSFAC 6.0-based Patch 1.0 algorithm. ( h ) Human MMP14 promoter activity was analyzed in astrocytes transfected with serial deletion reporter constructs (-1500, -1000, -500, -300, -200, and -100 bp) for 48 h. Data represent three independent experiments (duplicates per experiment). Significantly different at **, p < 0.01; ***, p < 0.001. ( i ) Ex p erimental design for NFATc2 cotransfection with MMP14 reporter constructs in human astrocytes for 24 h, followed by luciferase activity measurement. ( j ) The change in luciferase activity (Δ luciferase activity) for the MMP14 promoter deletion construct (-200Δ) was significantly reduced compared with that of the full MMP14 promoter (-200 bp). Significantly different at *, p < 0.05. ( k ) Western blot analysis showing the MMP14 p rotein levels in astrocytes treated with increasing doses of NFATc2 . ( l ) Densitometry analysis of the MMP14 protein levels shown in panel ( k ). The data represent four independent experiments. One-way ANOVA (*, p < 0.05; **, p < 0.01)

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Expressing, In Vitro, Western Blot, Staining, Transfection, Over Expression, Binding Assay, Activity Assay, Construct, Cotransfection, Luciferase

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: Phytoestrogen antagonizes NFATc2 transcriptional activity. ( a ) Experimental design for MMP14 promoter transfection into human astrocytes under normal (HTT-25Q) or HD (mHTT-103Q) conditions, followed by genistein treatment and a luciferase reporter assay. (b) Genistein significantly reduced mHTT (103Q)-induced MMP14 promoter activity (–200 bp construct). The data represent the means of five independent experiments. The error bars represent the means ± SEMs. One-way ANOVA (*, p < 0.05; ***, p < 0.001). (c) Experimental design for MMP14 promoter transfection followed by treatment with genistein, fulvestrant (ER inhibitor), or cyclosporin A (NFAT inhibitor) under normal and HD conditions, with a subsequent luciferase assay. (d) Cyclosporin A decreased mHTT (103Q)-induced MMP14 promoter activity (–200 bp), whereas fulvestrant blocked the genistein-mediated reduction in MMP14 promoter activity under HD conditions. The data represent the means of three independent experiments. One-way ANOVA (*, p < 0.05). (e) Western blot analysis of human astrocytes cotransfected with NFATc2 and treated with genistein for 24 h. (f) Densitometry analysis of the MMP14 protein levels shown in panel (e). The data represent four independent experiments. One-way ANOVA (*, p < 0.05; **, p < 0.01). (g) Experimental design for mammalian two-hybrid interactions between NFATc2 and ER α . The full-length (amino acids 1 to 927), regulatory (amino acids 1 to 407), DNA-binding (amino acids 407 to 677), and C-terminal (amino acids 677 to 927) domains of NFATc2 were fused to the Gal4 DNA-binding domain (GAL4-NFATc2). ER α was fused to the VP16 transcriptional activation domain (VP16-ER α ). (h) GAL4-NFATc2 alone increased luciferase activity, whereas cotransfection with VP16-ERα significantly reduced GAL4-NFATc2-induced luciferase activity. The data represent three independent experiments. **, p < 0.01; ***, p < 0.001. (i) VP16-ERα reduced luciferase activity in a dose-dependent manner. The data represent three independent experiments. *, p < 0.05. (j) NFATc2-△1 resulted in reduced luciferase activity upon cotransfection with VP16-ER. The data represent three independent experiments. *, p < 0.05. (k) Experimental workflow for immunoprecipitation (IP) analysis to detect NFATc2-ERα protein‒protein interactions. (l) ERα and NFATc2 interaction was verified by IP followed by Western blotting. (m) Proposed model illustrating the inhibition of the WNT5B‒NFATc2‒MMP14 axis by the genistein‒ERα interaction. WNT5B activates noncanonical WNT signaling, leading to NFATc2 and the induction of MMP14 transcription. Genistein activates ERα, which physically interacts with NFATc2 and antagonizes its transcriptional activity. Schematic created with BioRender.com

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Construct, Western Blot, Binding Assay, Activation Assay, Cotransfection, Immunoprecipitation, Inhibition

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: WNT5B overexpression enhances MMP14 expression in striatal astrocytes, leading to extracellular matrix (ECM) disruption and neuronal atrophy in HD mice. ( a ) Experimental design for AAV (pAAV-GFAP(pro)-Control-mCherry or pAAV-GFAP(pro)-WNT5B-P2A-mCherry) delivery into the dorsal striatum of N171-82Q mice via bilateral stereotaxic injection. ( b ) Brain sections stained with cresyl violet. Scale bars: 20 μm. Right: quantification of neuronal size in the dorsal striatum. A total of 40 cells per group were analyzed (8 cells per mouse; N = 5 mice per group: WT, N171-82Q, and N171-82Q + WNT5B). The error bars represent the means ± SEMs. One-way ANOVA ( ***, p < 0.001 ). ( c ) DARPP-32 immunoreactivity was reduced in the striatum of N171-82Q mice and further decreased in N171-82Q + WNT5B mice. Nuclei were counterstained with DAPI. Scale bars: 20 μm. ( d ) Quantitation of DARPP-32 immunoreactivity. A total of 20 cells per group were analyzed (4 cells per mouse; N = 5 mice per group: WT, N171-82Q, and N171-82Q + WNT5B). *, p < 0.05; **, p < 0.01; ***, p < 0.001. ( e ) Immunostaining for MMP14 and GFAP. Three-dimensional (3D) images were reconstructed for Sholl analysis of GFAP-positive astrocytes in the three groups of mice. Scale bars: 20 μm. ( f ) Quantification of MMP14 immunoreactivity in GFAP-positive cells in the dorsal striatum. A total of 15 cells per group were analyzed (3 cells per mouse; N = 5 mice per group: WT, N171-82Q, and N171-82Q + WNT5B). *, p < 0.05; ***, p < 0.001. ( g ) Quantification of the total number of processes associated with astrocytic processes via Sholl analysis. A total of 15 cells per group were analyzed (3 cells per mouse; N = 5 mice per group). One-way ANOVA (*, p < 0.05; **, p < 0.01; ***, p < 0.001). ( h ) Western blot analysis of the MMP14 and GFAP protein levels in striatal lysates from WT, N171-82Q, and N171-82Q + WNT5B mice. ( i ) Densitometry of MMP14 and GFAP protein levels from panel H. N = 3 samples per group. One-way ANOVA (*, p < 0.05; **, p < 0.01). ( j ) Alcian blue staining revealed the loss of glycosaminoglycans in the dorsal striatum of N171-82Q + WNT5B mice. Scale bar: 20 μm. Right: quantification of Alcian blue staining density in the dorsal striatum. Three ROIs (0.05 mm 2 each) per mouse were analyzed ( n = 5 mice per group). *, p < 0.05; ***, p < 0.001. ( k ) mHTT (mEM48) immunoreactivity was elevated in the striatum of N171-82Q + WNT5B mice. Scale bars: 10 μm. Right: quantification of mHTT immunoreactivity. A total of 25 cells per group were analyzed (5 cells per mouse; N = 5 mice per group). *, p < 0.05; ***, p < 0.001

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Over Expression, Expressing, Disruption, Control, Injection, Staining, Quantitation Assay, Immunostaining, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: WNT5B gain of function exacerbates motor deficits and shortens the lifespan of HD mice. ( a ) Experimental design for the delivery of pAAV-GFAP(pro)-Control-mCherry or pAAV-GFAP(pro)-WNT5B-P2A-mCherry viruses into the dorsal striatum of N171-82Q mice via bilateral stereotaxic injection, followed by behavioral testing two weeks post-injection. (b) Latency to fall was reduced in N171-82Q + WNT5B mice during the rotarod test ( n = 5 mice per group: WT, N171-82Q, and N171-82Q + WNT5B). The error bars represent the means ± SEMs. One-way ANOVA (Trial #1, P = 0.812; Trial #2, P = 0.129; Trial #3, *, p < 0.05). (c) The number of forelimbs and hindlimbs clasping during the tail suspension test at 2 weeks post-injection. N = 5 (WT, N 171-82Q, N171-82Q + WNT5B). *, p < 0.05; ***, p < 0.001. (d) Experimental design for the delivery of AAV-HTT(25Q) (control) or AAV-HTT(103Q) (HD model) into the dorsal striatum of WT mice. For astrocyte-specific WNT5B overexpression, either pAAV-GFAP(pro)-Control-mCherry or pAAV-GFAP(pro)-WNT5B-P2A-mCherry was coinjected. Behavioral testing was conducted three weeks post-injection. (e) Rotarod test showing a reduced latency to fall in 103Q mice compared with 25Q controls, with the greatest impairment observed in 103Q + WNT5B mice (25Q, N = 9; 103Q, N = 10; 103Q + WNT5B, N = 9). One-way ANOVA (significantly different from 25Q mice at **, p < 0.01 and different from 103Q at # , p < 0.05). (f) Tail suspension test at 2 weeks post-injection showing increased forelimb and hindlimb clasping in 103Q and 103Q + WNT5B mice (25Q, N = 9; 103Q, N = 10; 103Q + WNT5B, N = 9). *, p < 0.05; **, p < 0.01. (g) Kaplan‒Meier survival analysis showing that WNT5B overexpression significantly reduced the life span of N171-82Q mice ( n = 7 mice per group: WT, N171-82Q, and N171-82Q + WNT5B). (h) Body weights of N171-82Q + WNT5B mice compared with those of N171-82Q and WT mice ( n = 7 mice per group)

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Control, Injection, Suspension, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: Astrocyte-specific knockdown of Wnt5b, Nfatc2 , or Mmp14 rescues ECM degradation and neuropathology in HD mice. ( a ) Experimental design for astrocyte-specific knockdown of Wnt5b , Nfatc2 , or Mmp14 with AAV-GFAP(pro)-Cre and Cre-dependent shRNA in the dorsal striatum of AAV-HTT (25Q) or AAV-mHTT (103Q) mice, followed by neuropathological assessment four weeks post-injection. (b) Representative brain sections confirming accurate delivery of AAVs into the dorsal striatum. (c) Cresyl violet (Nissl) staining of brain sections. Scale bars: 20 μm. Lower panel: quantification of neuronal size in the dorsal striatum. A total of 30 cells per group were analyzed (6 cells per mouse; N = 5 mice per group: 25Q-shCont, 103Q-shCont, 103Q-sh Wnt5b , 103Q-sh Nftac2 , and 103Q-sh Mmp14 ). One-way ANOVA (**, p < 0.01; ***, p < 0.001). (d) Immunostaining and quantification of DARPP-32 immunoreactivity. Scale bars: 20 μm. A total of 30 cells per group were analyzed (6 cells per mouse; N = 5 mice per group (25Q-shCont, 103Q-shCont, 103Q-shWnt5b, 103Q-shNftac2, 103Q-shMmp14)). ***, p < 0.001. (e) Immunostaining and quantification of MMP14 intensity in GFAP-positive astrocytes. Scale bar: 20 μm. A total of 30 cells per group were analyzed (6 cells per mouse; N = 5 mice per group). ***, p < 0.001. (f) Alcian blue staining of the striatum showing the preservation of glycosaminoglycans following the knockdown of Wnt5b , Nfatc2 , or Mmp14 . Scale bars: 20 μm. Lower panel: quantification of Alcian blue density in the dorsal striatum. Six ROIs (0.05 mm 2 each) per mouse were analyzed ( n = 5 mice per group). ***, p < 0.001

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Knockdown, shRNA, Injection, Staining, Immunostaining, Preserving

Journal: Signal Transduction and Targeted Therapy

Article Title: Astrocytic noncanonical WNT5B signaling modulates extracellular matrix remodeling and neuropathology in Huntington’s disease

doi: 10.1038/s41392-025-02545-9

Figure Lengend Snippet: Genistein ameliorates motor deficits and extends lifespan in HD mice. ( a ) Experimental design for the behavioral assessment of N171-82Q mice treated with genistein or saline via I.P. injection from postnatal days 30 to 90. (b) Forelimb and hindlimb clasping behavior was quantified in the tail suspension test (WT, N = 7; N171-82Q, N = 4; N171-82Q + genistein, N = 6). One-way ANOVA (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (c) Representative still images from the accelerated wheel test (left) and computer-assisted footprint analysis (right) in the wheel running paradigm across three experimental groups. (d) Genistein treatment improved stride length and stride width in N171-82Q (WT, N = 7; N171-82Q, N = 4; N171-82Q + genistein, N = 7). One-way ANOVA (***, p < 0.001). The data are presented as the means ± SEMs. (e) Experimental design for behavioral tests in AAV-mHTT (103Q)-induced HD mice following one month of i.p. administration of genistein or saline. (f) Rotarod performance showing that genistein significantly restored the latency to fall in 103Q mice relative to that in 103Q vehicle control mice (25Q, N = 10; 103Q, N = 9; 103Q + genistein, N = 10). One-way ANOVA (*, p < 0.05). (g) Tail suspension test showing that genistein alleviated forelimb and hindlimb clasping in 103Q mice compared with 103Q vehicle controls (25Q, N = 10; 103Q, N = 9; 103Q + genistein, N = 10). *, p < 0.05; **, p < 0.01; ***, p < 0.001. (h) Kaplan‒Meier survival analysis showing that genistein significantly prolonged the lifespan of N171-82Q mice ( n = 10 mice per group: N171-82Q and N171-82Q + genistein). (i) Genistein treatment improved body weight maintenance in N171-82Q mice starting at 20 weeks of age. (j) Proposed model illustrating therapeutic modulation of the WNT5B-NFATc2-MMP14 signaling axis. Upregulation of WNT5B by mHTT activates noncanonical WNT signaling via NFATc2, inducing MMP14 transcription in reactive astrocytes. Elevated MMP14 leads to ECM disruption, mHTT aggregation, and MSN damage. Genistein antagonizes NFATc2 activity and suppresses MMP14 expression, thereby mitigating ECM degradation and neurodegeneration in HD. Schematic created with BioRender.com

Article Snippet: Primary antibody incubation was performed overnight (24 h) at 4 °C, using the following antibodies at the indicated dilutions: WNT5B (1:100, Santa Cruz, USA), MMP14 (1:200, Abcam, USA), NFATC2 (1:100, Invitrogen, USA), S100β (1:200, Synaptic Systems, Germany), β-catenin (1:200, Santa Cruz, USA), and non-phospho (active) β-catenin (Ser33/37/Thr41; 1:1000, Cell Signaling, USA).

Techniques: Saline, Injection, Suspension, Control, Disruption, Activity Assay, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: The SCD5 Gene Modulates Adipogenic Differentiation via the WNT5B Signaling Pathway in Xinjiang Brown Cattle

doi: 10.3390/ani15243547

Figure Lengend Snippet: The SCD5 Regulates Adipogenesis Through the WNT5B Signaling Pathway. ( a ) WNT5B interference efficiency 48 h post-transfection. siRNA2 was selected for subsequent experiments ( n = 3). ( b ) β-catenin mRNA expression after WNT5B silencing ( n = 3). ( c ) Protein levels of WNT5B , β-catenin , and phosphorylated β-catenin ( p-β-catenin ) as determined by Western blot 48 h post-transfection ( n = 3). ( d ) Cell proliferation (CCK-8) 48 h post-transfection ( n = 3). ( e ) mRNA expression of differentiation markers at 24 h and 8 d. ( f ) Lipid accumulation by Oil Red O staining at day 8. Quantification at 510 nm after extraction and the OD value represents the quantified intracellular triglyceride (TG) content. All data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After blocking, the membrane was incubated overnight at 4 °C with the primary antibody prepared in a specialized dilution buffer: GAPDH (1:5000, Proteintech, Wuhan, China, cat #10494-1-AP), CDK1 (1:2000, Proteintech, Wuhan, China, cat #19532-1-AP), CDK6 (1:1000, Proteintech, Wuhan, China, cat #14052-1-AP), PPARγ (1:1000, Proteintech, Wuhan, China, cat #16643-1-AP), C/EBPα (1:1000, Proteintech, Wuhan, China, cat #29388-1-AP), SCD5 (1:1000, Aviva Systems Biology, San Diego, CA, USA, cat #ARP65045_P050), WNT5B (1:1000, biorbyt, Cambridge, UK, cat #orb577989), β-Catenin (1:1000, Abcam, Cambridge, UK, cat #ab32572), and p-β-Catenin (1:500, Affbiotech, Beijing, China, cat #affbiotech).

Techniques: Transfection, Expressing, Western Blot, CCK-8 Assay, Staining, Extraction