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human recombinant protein wnt5a (R&D Systems)

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R&D Systems human recombinant protein wnt5a
Human Recombinant Protein Wnt5a, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/wnt5a+proteins/Recombinant+Human%2FMouse+Wnt-5a+Protein%2C+CF/pm41791386-192-26-30
Average 94 stars, based on 117 article reviews

human recombinant protein wnt5a - by Bioz Stars, 2026-10

94/100 stars

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Expressing:

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.
Article Snippet: .. Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer. .. Unincorporated fluorescein was removed by fluorescentdye-removal columns (Thermo Fisher Scientific Inc.) TMEM67-Nt protein (encoding amino acids F39-T478, predicted molecular mass 48 kDa) was expressed following transfection of HEK293 cells with pSec2A constructs (Life Technologies Ltd) using conditions recommended by the manufacturer.

In Vitro:

Binding Assay:

Purification:

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway
Article Snippet: .. Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer. .. Unincorporated fluorescein was removed by fluorescent-dye-removal columns (Thermo Fisher Scientific Inc.) TMEM67-Nt protein (encoding amino acids F39-T478, predicted molecular mass 48 kDa) was expressed following transfection of HEK293 cells with pSec2A constructs (Life Technologies Ltd) using conditions recommended by the manufacturer.

Recombinant:

Article Title: Wnt5a is required for cardiac outflow tract septation in mice.
Article Snippet: To assess dynamic changes in cytosolic Ca2 concentration ([Ca2 ]i), we used the Ca 2 -sensitive fluorophore fura-2 AM (Molecular Probes) as previously published (31,32). .. To determine the effects of the recombinant mouse Wnt3a and Wnt5a proteins (R&D Systems), along with human endothelin-1 (Et-1, Sigma Chemical Co.) on CNC cell [Ca2 ]i, baseline measurements were obtained for 5 min before the substances were superfused over the cells. ..

Article Title: Wnt11 in regulation of physiological and pathological cardiac growth
Article Snippet: The experiments were started 2 days after isolation in complete serum- free medium (CSFM) containing 2.5 mg/ml bovine serum albumin (BSA) (Sigma), 5 μg/ml– 5 μg/ml– 5 ng/ml insulin– transferrin– sodium selenite (ITS) (Roche, Basel, Switzerland), 2.8 mM Napyruvate, 0.1 nM T3 (Sigma), and 1% PS in DMEM/F12. .. Recombinant Wnt11 and Wnt5a proteins (R&D Systems, Minneapolis, MN, USA) were added as 200 ng/ ml. ..

Article Title: Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions.
Article Snippet: .. Recombinant Wnt3a and Wnt5a proteins were purchased from R & D Systems (Minneapolis, MN, USA). .. Wnt1 was purchased from Peprotech, USA.

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway
Article Snippet: .. Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer. .. Unincorporated fluorescein was removed by fluorescent-dye-removal columns (Thermo Fisher Scientific Inc.) TMEM67-Nt protein (encoding amino acids F39-T478, predicted molecular mass 48 kDa) was expressed following transfection of HEK293 cells with pSec2A constructs (Life Technologies Ltd) using conditions recommended by the manufacturer.

Negative Control:

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway
Article Snippet: .. Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer. .. Unincorporated fluorescein was removed by fluorescent-dye-removal columns (Thermo Fisher Scientific Inc.) TMEM67-Nt protein (encoding amino acids F39-T478, predicted molecular mass 48 kDa) was expressed following transfection of HEK293 cells with pSec2A constructs (Life Technologies Ltd) using conditions recommended by the manufacturer.

Image Search Results

a RNA-seq was performed in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3), with a threshold of |FC| > 1.5 and adjusted P-value < 0.05. KEGG enrichment analysis of genes upregulated in Adipoq Cre ; Ctnnb1 flox/flox mice relative to control mice. b mRNA expression of Wnt5a and Fzd5 in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). c Immunoblotting for Wnt5a, pCaMKK2, CaMKK2, pAMPK, AMPK, Atgl, Sirt1, Pgc1α in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). d Wnt5a and Fzd5 expression in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). e Immunoblotting for Wnt5a, pCaMKII, CaMKII, pCaMKK2, CaMKK2, pAMPK, AMPK in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). f mRNA expression of triglyceride lipolysis genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 5–6). g mRNA expression of fatty acid β-oxidation genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 6). h Immunoblotting for Cpt1, Atgl in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). i Immunoblotting for Sirt1, Pgc1α, Pparγ, Pparɑ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). j mRNA expression of Pparγ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 7). k Schematic summary of β-catenin suppression results in activation of Wnt/Ca 2+ -AMPK axis, highlighting non-canonical pathways involved in the thermogenic response. The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were one-way ANOVA with Tukey’s correction for multiple comparisons or unpaired two-tailed t -tests.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a RNA-seq was performed in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3), with a threshold of |FC| > 1.5 and adjusted P-value < 0.05. KEGG enrichment analysis of genes upregulated in Adipoq Cre ; Ctnnb1 flox/flox mice relative to control mice. b mRNA expression of Wnt5a and Fzd5 in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). c Immunoblotting for Wnt5a, pCaMKK2, CaMKK2, pAMPK, AMPK, Atgl, Sirt1, Pgc1α in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). d Wnt5a and Fzd5 expression in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). e Immunoblotting for Wnt5a, pCaMKII, CaMKII, pCaMKK2, CaMKK2, pAMPK, AMPK in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). f mRNA expression of triglyceride lipolysis genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 5–6). g mRNA expression of fatty acid β-oxidation genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 6). h Immunoblotting for Cpt1, Atgl in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). i Immunoblotting for Sirt1, Pgc1α, Pparγ, Pparɑ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). j mRNA expression of Pparγ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 7). k Schematic summary of β-catenin suppression results in activation of Wnt/Ca 2+ -AMPK axis, highlighting non-canonical pathways involved in the thermogenic response. The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were one-way ANOVA with Tukey’s correction for multiple comparisons or unpaired two-tailed t -tests.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Activation Assay, Two Tailed Test

a Experimental strategy to suppress β-catenin in vitro by employing adipocytes differentiated from SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), MSAB, Rosiglitazone, or MSAB plus Rosiglitazone ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to suppress β-catenin while inhibiting the Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of Rosiglitazone-differentiated adipocytes treated with vehicle (0.1% DMSO), MSAB, or MSAB plus BAPTA-AM ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots and measured maximal respiration levels across the three treatment groups (Vehicle, MSAB, MSAB and BAPTA-AM) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests or two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin in vitro by employing adipocytes differentiated from SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), MSAB, Rosiglitazone, or MSAB plus Rosiglitazone ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to suppress β-catenin while inhibiting the Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of Rosiglitazone-differentiated adipocytes treated with vehicle (0.1% DMSO), MSAB, or MSAB plus BAPTA-AM ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots and measured maximal respiration levels across the three treatment groups (Vehicle, MSAB, MSAB and BAPTA-AM) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests or two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot

a Experimental strategy to suppress Wnt5a/Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Box5, MSAB, or MSAB plus Box5 ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to supply exogenous Wnt5a recombinant protein in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Wnt5a recombinant protein, MSAB, or MSAB plus Wnt5a recombinant protein ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots, measured basal respiration and measured maximal respiration levels in cultured peri-weaning adipocytes across the six treatment groups (Vehicle, Wnt5a recombinant protein, MSAB, MSAB plus Wnt5a recombinant protein, Box5, MSAB plus Box5) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress Wnt5a/Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Box5, MSAB, or MSAB plus Box5 ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to supply exogenous Wnt5a recombinant protein in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Wnt5a recombinant protein, MSAB, or MSAB plus Wnt5a recombinant protein ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots, measured basal respiration and measured maximal respiration levels in cultured peri-weaning adipocytes across the six treatment groups (Vehicle, Wnt5a recombinant protein, MSAB, MSAB plus Wnt5a recombinant protein, Box5, MSAB plus Box5) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot, Recombinant, Cell Culture

a Experimental strategy to suppress β-catenin using adipocytes differentiated from human subcutaneous lipoaspirates. b Microscopy images of human subcutaneous adipocytes following treatment with vehicle (0.1% DMSO) and MSAB after maturation ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 30 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 30 cells). c mRNA expression of adipogenic genes in ( b ) ( n = 3 biological replicates). d mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). e Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis, Wnt/β-catenin signaling pathway and thermogenic protein in ( b ) ( n = 3 biological replicates). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin using adipocytes differentiated from human subcutaneous lipoaspirates. b Microscopy images of human subcutaneous adipocytes following treatment with vehicle (0.1% DMSO) and MSAB after maturation ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 30 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 30 cells). c mRNA expression of adipogenic genes in ( b ) ( n = 3 biological replicates). d mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). e Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis, Wnt/β-catenin signaling pathway and thermogenic protein in ( b ) ( n = 3 biological replicates). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests.

Techniques: Microscopy, Staining, Labeling, Expressing, Western Blot

( A ) UMAP of epithelial subtypes in control and NEC mice. ( B ) Representative immunofluorescence images for Lyz (red), Epacm (green) and DAPI (blue) on paraffin-embedded distal ileum from indicated group in experiment 3. White arrow indicates the Lyz stained PCs, Scale bar: 10 μm. ( C , D ) The number of Lyz + PCs in B ( C ) and mRNA levels of Lyz ( D ) were compared among different groups. ( E ) Violin plots showed the expression of Wnt/PCP pathway genes in mouse PCs subsets revealed by scRNA-seq analysis. ( F ) The mRNA level of Damm1 was compared in IECs isolated from different groups. ( G ) Organoids derived from mice were cultured in medium with DMSO, FEX (5 μm), or FEX (10 μm) from day 1. The organoids were collected and stained for Lyz on day 7. Arrowheads point at PCs (Lyz + , in pink), scale bars: 50 μm. ( H ) qRT-PCR analysis of Daam1 in organoids cultured with DMSO, FEX (5 μm), or FEX (10 μm). ( I ) Representative organoid formation images on day 7 in different groups supplemented with ENR, Wnt5a-containing ENR, and Wnt5a-containing ENR + FEX. Representative marker genes for PCs (Lyz, green) cells are highlighted by fluorescent reporters. Arrowheads point at PCs (Lyz + , in green), scale bars: 50 μm. All statistical data are shown as the means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: NPJ Biofilms and Microbiomes

Article Title: Disruption of bile acid homeostasis potentiates Paneth cell ablation by activating the intestinal Farnesoid X receptor in necrotizing enterocolitis

doi: 10.1038/s41522-025-00904-6

Figure Lengend Snippet: ( A ) UMAP of epithelial subtypes in control and NEC mice. ( B ) Representative immunofluorescence images for Lyz (red), Epacm (green) and DAPI (blue) on paraffin-embedded distal ileum from indicated group in experiment 3. White arrow indicates the Lyz stained PCs, Scale bar: 10 μm. ( C , D ) The number of Lyz + PCs in B ( C ) and mRNA levels of Lyz ( D ) were compared among different groups. ( E ) Violin plots showed the expression of Wnt/PCP pathway genes in mouse PCs subsets revealed by scRNA-seq analysis. ( F ) The mRNA level of Damm1 was compared in IECs isolated from different groups. ( G ) Organoids derived from mice were cultured in medium with DMSO, FEX (5 μm), or FEX (10 μm) from day 1. The organoids were collected and stained for Lyz on day 7. Arrowheads point at PCs (Lyz + , in pink), scale bars: 50 μm. ( H ) qRT-PCR analysis of Daam1 in organoids cultured with DMSO, FEX (5 μm), or FEX (10 μm). ( I ) Representative organoid formation images on day 7 in different groups supplemented with ENR, Wnt5a-containing ENR, and Wnt5a-containing ENR + FEX. Representative marker genes for PCs (Lyz, green) cells are highlighted by fluorescent reporters. Arrowheads point at PCs (Lyz + , in green), scale bars: 50 μm. All statistical data are shown as the means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For experiments involving Wnt5a (645-WN-010/CF, R&D Systems) ligands, the media was made by supplementing ENR media with 100 ng/ml Wnt5a.

Techniques: Control, Immunofluorescence, Staining, Expressing, Isolation, Derivative Assay, Cell Culture, Quantitative RT-PCR, Marker

a General schematic of the TetON inducible gene expression system used in this study. In the absence of doxycycline, the reverse tetracycline-controlled transactivator (rtTA) is inactive. Upon doxycycline addition, rtTA binds to the tetO promoter and activates transcription of the gene of interest, leading to protein expression. b Doxycycline-induced overexpression of HA-tagged PRICKLE3 in HEK T-REx 293 PRICKLE3 TetON cells and of PRICKLE1 in the corresponding inducible cell line. c Induction of HA-PRICKLE3 expression by doxycycline in HEK T-REx 293 PRICKLE3 TetON cells. HEK T-REx 293 wildtype (WT) cells served as a control for doxycycline effects. Arrowheads indicate phosphorylation-dependent shifts in the electrophoretic mobility of VANGL proteins. α-TUBULIN was used as a loading control. Representative result from n = 5. d , e Densitometric quantification of Western blot signals. Values were normalized to untreated cells. Statistical analysis was performed using an unpaired t -test; corresponding p -values are shown ( n = 5). f Effect of recombinant WNT stimulation on VANGL phosphorylation. HEK T-REx 293 PRICKLE3 TetON cells were pre-treated overnight with the porcupine inhibitor LGK-974 to block endogenous WNT ligand secretion and subsequently stimulated with 100 ng/ml human recombinant WNT5A or WNT3A for 3 h. Arrowheads indicate phosphorylation-dependent mobility shifts of ROR. α-TUBULIN served as a loading control. Representative result from n = 4. g , h Densitometric quantification of Western blot signals from panel f. Data were normalized to untreated controls. Statistical analysis was performed using an unpaired t -test; corresponding p -values are reported ( n = 4).

Journal: Communications Biology

Article Title: PRICKLE3 protects VANGL proteins from CK1-mediated phosphorylation and RNF43-mediated degradation

doi: 10.1038/s42003-025-09422-9

Figure Lengend Snippet: a General schematic of the TetON inducible gene expression system used in this study. In the absence of doxycycline, the reverse tetracycline-controlled transactivator (rtTA) is inactive. Upon doxycycline addition, rtTA binds to the tetO promoter and activates transcription of the gene of interest, leading to protein expression. b Doxycycline-induced overexpression of HA-tagged PRICKLE3 in HEK T-REx 293 PRICKLE3 TetON cells and of PRICKLE1 in the corresponding inducible cell line. c Induction of HA-PRICKLE3 expression by doxycycline in HEK T-REx 293 PRICKLE3 TetON cells. HEK T-REx 293 wildtype (WT) cells served as a control for doxycycline effects. Arrowheads indicate phosphorylation-dependent shifts in the electrophoretic mobility of VANGL proteins. α-TUBULIN was used as a loading control. Representative result from n = 5. d , e Densitometric quantification of Western blot signals. Values were normalized to untreated cells. Statistical analysis was performed using an unpaired t -test; corresponding p -values are shown ( n = 5). f Effect of recombinant WNT stimulation on VANGL phosphorylation. HEK T-REx 293 PRICKLE3 TetON cells were pre-treated overnight with the porcupine inhibitor LGK-974 to block endogenous WNT ligand secretion and subsequently stimulated with 100 ng/ml human recombinant WNT5A or WNT3A for 3 h. Arrowheads indicate phosphorylation-dependent mobility shifts of ROR. α-TUBULIN served as a loading control. Representative result from n = 4. g , h Densitometric quantification of Western blot signals from panel f. Data were normalized to untreated controls. Statistical analysis was performed using an unpaired t -test; corresponding p -values are reported ( n = 4).

Article Snippet: Following this, non-canonical WNT signaling was activated by incubating the cells with recombinant human WNT5A (645-WN, R&D Systems) at a concentration of 100 ng/mL for 3 h. Similarly, canonical WNT signaling was activated by incubating the cells with 100 ng/mL of recombinant human WNT3A (5036-WN, R&D Systems) for 3 h. To assess protein degradation kinetics, cells were pretreated with doxycycline overnight.

Techniques: Gene Expression, Expressing, Over Expression, Control, Phospho-proteomics, Western Blot, Recombinant, Blocking Assay

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