phosphorylated β catenin  (Proteintech)

Journal: Advanced Science

Article Title: Targeting Fibrotic Scarring by Mechanoregulation of Il11ra1 + /Itga11 + Fibroblast Patterning Promotes Axon Growth after Spinal Cord Injury

doi: 10.1002/advs.202513476

Figure Lengend Snippet: Mechanotransduction induced the Itga11 + /Il11ra1 + fibroblast subset via LRP6‐Wnt/β‐Catenin‐MMP7 signaling axis. A–C) Representative chopped Western blot images A) and quantitation of the expression of phosphorylated β‐catenin B) and β‐catenin C) in vivo. N = 3 samples, respectively. D–F) Representative chopped Western blot images D) and quantitation of the expression of phosphorylated β‐catenin E), and β‐catenin F) in vitro. N = 3 samples, respectively. G) Representative multichannel immunostaining images revealing the expression active β‐catenin and phalloidin in the migrating fibroblasts in different groups. The white boxed regions were the high magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. H)The quantifications of the percentage of active β‐catenin entering nucleus. N = 9 cells. I) Heatmap showing the expression levels of LRP5 and LRP6 from scRNA‐seq. J,K) The representative chopped Western blot images J) and quantitation K) showing the expression of LRP6 and phosphorylated LRP6 proteins in vivo. N = 3 animals. L) Schematic of the experimental design for the in vitro validation. M) Western blot results of related proteins expressions in vitro following intervention with HLY78 and Salinomycin respectively. N) Representative multichannel immunostaining images showing the expression active β‐catenin and phalloidin in the migrating fibroblasts in Medium hydrogel group following intervention with HLY78. The white boxed regions were higher magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. The quantifications of the proportion of active β‐catenin entering nucleus following the intervention with HLY78 were listed on the right. N = 9 cells. O,P) Representative multichannel immunostaining images showing the expression active β‐catenin and phalloidin in the migrating fibroblasts in High hydrogel group O) and Low hydrogel group P) following intervention with Salinomycin. The white boxed regions were higher magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. The quantifications of the proportion of active β‐catenin entering nucleus following the intervention with HLY78 were listed on the right. N = 9 cells, respectively. Q) Flow cytometric analysis of Itgall + Il11ra1 + fibroblasts in vitro in Medium hydrogel group following intervention with HLY78. N = 3 samples. R,S) Flow cytometric analysis of Itgall + Il11ra1 + fibroblasts in vitro in High hydrogel group R) and Low hydrogel group S) following intervention with Salinomycin. N = 3 samples, respectively. T) Representative chopped Western blot images showing the expression of the related proteins in vivo following intervention with HLY78 and Salinomycin respectively. U) Schematic representation of the LRP6‐Wnt/β‐Catenin‐MMP7 pathway axis. Data were shown as the mean ± SD; p values were determined by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test or two‐tailed paired t ‐tests.

Article Snippet: To assess the role of β‐Catenin phosphorylation, fibroblasts seeded onto hydrogels were treated with β‐Catenin phosphorylation inhibitor Laduviglusib (10 μM, HY‐10182, MedChem‐Express, USA) or β‐Catenin phosphorylation enhancer (E)‐Ferulic acid (50 μ m , HY‐N0060B, MedChem‐Express, USA) in the medium.

Techniques: Western Blot, Quantitation Assay, Expressing, In Vivo, In Vitro, Immunostaining, Biomarker Discovery, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: FCN3 regulated Wnt/β-catenin signaling to influence Treg activation. ( A - B ) qRT-PCR and WB analysis of APC and β-catenin in HCC tumor and adjacent non-tumor tissues. ( C ) Clinical sample IF staining showing β-catenin expression and localization with DAPI nuclear counterstain. ( D - E ) The levels of APC and β-catenin in HCC cell lines were assessed by qRT-PCR and WB. ( F - G ) qRT-PCR and WB evaluation of APC and β-catenin in Hep3B cells treated with OE-NC, OE-FCN3, OE-FCN3 + DMSO, or OE-FCN3 + LY2090314 (β-catenin activator). ( H ) The levels of TGF-β1 and IL-10 in cell culture supernatant of each group were examined by ELISA. ( I - M ) Wound healing, Transwell and flow cytometry assays evaluating the effect of OE-FCN3 and OE-FCN3 + LY2090314 on cell migration, invasion and apoptosis of Hep3B cells. ( N ) Flow cytometry was performed to analyze the effects of OE-FCN3 and OE-FCN3 + LY2090314 treatment in Hep3B cells on the proportion of CD4⁺CD25⁺FOXP3 + Treg cells in Hep3B-PBMC co-cultures. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. N/ THLE-2/ OE-NC/ OE-FCN3 + DMSO

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against FCN3 (#ABIN521997, 1:2000; Antibodies Online, Aachen, Germany), FOXP3 (#22228-1-AP, 1:500; Proteintech), CD25 (#83896-1-RR, 1:1000; Proteintech), APC (#sc-9998, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (#66379-1-Ig, 1:5000; Proteintech), phosphorylated β-catenin (p-β-catenin) (#80067-1-RR, 1:1000; Proteintech) or STT3A (#ab320831, 1:20000; Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: N-glycosylated FCN3 influenced Wnt/β-catenin signaling and Treg activation. ( A ) qRT-PCR analysis of APC and β-catenin mRNA levels in Hep3B cells transfected with FCN3-WT or FCN3-N189Q. ( B ) WB analysis showing the levels of APC, β-catenin, and p-β-catenin. ( C ) Subcellular localization of β-catenin was analyzed by WB. ( D ) ELISA was conducted to assess the levels of TGF-β1 and IL-10 in cell culture supernatant of Hep3B cells transfected with FCN3-WT or FCN3-N189Q. ( E - K ) The effects of FCN3 glycosylation on cell viability, migration, invasion and apoptosis in Hep3B cells were respectively evaluated by CCK-8, wound healing, Transwell and flow cytometry assays. ( L ) Flow cytometry demonstrating the impact of FCN3 glycosylation on the proportion of CD4⁺CD25⁺FOXP3 + Treg cells in Hep3B-PBMC co-cultures. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. FCN3-WT

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against FCN3 (#ABIN521997, 1:2000; Antibodies Online, Aachen, Germany), FOXP3 (#22228-1-AP, 1:500; Proteintech), CD25 (#83896-1-RR, 1:1000; Proteintech), APC (#sc-9998, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (#66379-1-Ig, 1:5000; Proteintech), phosphorylated β-catenin (p-β-catenin) (#80067-1-RR, 1:1000; Proteintech) or STT3A (#ab320831, 1:20000; Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: STT3A involved in HCC progression via promoting FCN3 N-glycosylation. ( A ) Bioinformatics analysis revealing STT3A expression in normal and HCC liver tissues. ( B - D ) qRT-PCR, WB and IHC assays were performed to assess STT3A levels in HCC tumor and adjacent non-tumor tissues. ( E - F ) The mRNA and protein levels of STT3A in normal hepatocyte and HCC cell lines were analyzed by qRT-PCR and WB. ( G ) Correlation analysis between STT3A protein expression and FCN3 protein expression in tumor tissues from HCC patients. ( H ) Co-IP assay verified STT3A-FCN3 interaction. ( I ) WB analysis of FCN3 glycosylation in Hep3B cells transfected with sh-STT3A or sh-NC. ( J - K ) The effects of sh-STT3A on the expression of APC and β-catenin in Hep3B cells were examined by qRT-PCR and WB. ( L ) ELISA showing TGF-β1 and IL-10 levels in Hep3B cell supernatants following sh-STT3A or sh-NC treatment. ( M - Q ) Wound healing, Transwell and flow cytometry assays were conducted to evaluate the effects of sh-STT3A on Hep3B cell migration, invasion and apoptosis. ( R ) The influence of sh-STT3A on the proportion of CD4⁺CD25⁺FOXP3 + Treg cells in Hep3B-PBMC co-cultures was assessed by flow cytometry. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. N/ THLE-2/ sh-NC

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against FCN3 (#ABIN521997, 1:2000; Antibodies Online, Aachen, Germany), FOXP3 (#22228-1-AP, 1:500; Proteintech), CD25 (#83896-1-RR, 1:1000; Proteintech), APC (#sc-9998, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (#66379-1-Ig, 1:5000; Proteintech), phosphorylated β-catenin (p-β-catenin) (#80067-1-RR, 1:1000; Proteintech) or STT3A (#ab320831, 1:20000; Abcam, Cambridge, MA, USA).

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration