human recombinant protein wnt5a  (R&D Systems)

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

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