Journal: PLOS One

Article Title: Deciphering the synergistic mechanism of a novel flavonoid-antioxidant combination for asthma by combining systems pharmacology and experimental validation

doi: 10.1371/journal.pone.0346165

Figure Lengend Snippet: (A) . Transwell analysis the migrated cells after TGF-β1 (5ng/mL), 74DHF (100nM), and VC (500μM) treatment. Scale bar: 100μm. The quantification of the stained cells was shown on the right (n = 3). (B) . Real time PCR of EMT marker Acta2 and Cdh1 expression in BEAS-2B cells treated by TGF-β1 (5ng/mL), 74DHF (100nM) or VC (500μM) (n = 3). (C) WB analysis of α-SMA, CDH1, COL1A1 and AKT1 in BEAS-2B cells treated by TGF-β1 (5ng/mL), 74DHF (100nM) or VC (500μM) for 24 hours (n = 3). The grey intensity was quantified on the right. Data are presented as the means ± SEM compared by one-way ANOVA with Bonferroni's Multiple Comparison Test. (*, P < 0.05, **, P < 0.01, ***, P < 0.001).

Article Snippet: The membrane was incubated with primary antibody against α-SMA (Abcam, ab7817), CDH1 (CST, 1065), COL1A1 (CST, 3395), AKT1 (CST, 2938), and GAPDH (Abcam, ab70699) over night.

Techniques: Staining, Real-time Polymerase Chain Reaction, Marker, Expressing, Comparison

Journal: Journal of Biological Chemistry

Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function

doi: 10.1074/jbc.m110.190330

Figure Lengend Snippet: FIGURE 3. LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 m. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transducedwithlentiviralparticlesharboringshRNAagainstLrp4.ReductionofLrp4RNAlevelswasassessedbyreal-timequantitativePCR,priortotheaddition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

Article Snippet: Recombinant human DKK1 and LRP6 were purchased from R&D Systems.

Techniques: Inhibition, In Vitro, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Activity Assay

Journal: Pancreas

Article Title: Polarization of the Vacuolar Adenosine Triphosphatase Delineates a Transition to High-Grade Pancreatic Intraepithelial Neoplasm Lesions

doi: 10.1097/mpa.0000000000000201

Figure Lengend Snippet: FIGURE 4. The v-ATPase is necessary for normal Wnt/β-catenin signaling in primary PanIN cells. A, Phospho-LRP6 and total LRP6 levels increased with concanamycin exposure. B, Quantification of blots showing approximately 80% increase in phospho-LRP6 with concanamycin. C, Blockade of the v-ATPase led to diminished intracellular levels of β-catenin in primary PanIN cells compared with vehicle-treated cells. D, Immunoblots of active (nonphosphorylated) β-catenin show reduced levels with concanamycin 10 nM exposure despite increased representative blot from a total of 3 separate experiments.

Article Snippet: Antibodies to phospholow-density lipoprotein receptor-related protein-6 (LRP6) No. 1490; total LRP6, No. 2560; nonphosphorylated (active) β-catenin, No. 4270; total β-catenin, No. 8480; phosphoGSK3β, No. 9327; and total glycogen synthase kinase 3β (GSK3β) No. 9315 (Cell Signaling) were used to interrogate canonical Wnt/β-catenin signaling.

Techniques: Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells

doi: 10.1007/s12079-020-00551-w

Figure Lengend Snippet: Lipid rafts localization of LRP6 in SK-N-BE2 cells. a-e SK-N-BE2 cells, either untreated or treated with 10 nM tPA for 10 min at 37 °C, were lysed and the supernatant fraction was subjected to sucrose density gradient. After centrifugation, the gradient was fractionated and each fraction was recovered and analyzed by western blot using anti-LRP6 pAb (a), anti-phospho LRP6 pAb (p-LRP6) (b), anti-phospho β-catenin (p-β-catenin) pAb (c), anti-CD71 mAb (d) or an anti-flotillin pAb (e). Right panel: Densitometric analysis of sucrose gradient fractions. The columns indicate the percent distribution across the gel of raft-fractions 4–5 and 6 (Triton X-100-insoluble fractions) and 9–10 and 11 (Triton X-100-soluble fractions), as detected by densitometric scanning analysis. Results represent the mean ± SD from three independent experiments (*) p < 0.01 TX-100-insoluble fractions from tPA-treated cells vs TX-100-insoluble from control cells; (°) p < 0.01 TX-100-soluble fractions from tPA-treated cells vs TX-100-soluble from control cells. f SK-N-BE2 cells, untreated or treated with 10 nM tPA, were lysed in lysis buffer, followed by immunoprecipitation with goat anti-LRP6 pAb. An IgG isotypic control was employed. The immunoprecipitates were spotted onto nitrocellulose strips and incubated with Cholera Toxin B Subunit-Peroxidase (from Vibrio Cholerae), as described in Materials and Methods. As a control, the immunoprecipitates were assessed by Dot-blot with anti-LRP6 mAb. A representative experiment among three is shown. Bar graph in the right panel shows densitometric analysis. Results represent the mean ± SD from three independent experiments. (*) p < 0.01 tPA vs control cells

Article Snippet: The beads were removed by centrifugation (500×g for 1 min) and the supernatants were incubated with goat anti-LRP6 Ab (R&D Systems, Minneapolis, MI, USA) at 4 °C overnight.

Techniques: Centrifugation, Western Blot, Control, Lysis, Immunoprecipitation, Incubation, Dot Blot

Journal: Journal of Cell Communication and Signaling

Article Title: LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells

doi: 10.1007/s12079-020-00551-w

Figure Lengend Snippet: Effect of LRP1 silencing on LRP6 phosphorylation induced by tPA in SK- N BE2 cells. a SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with siRNA LRP1, were analyzed by Western blot, using anti-phospho LRP6 pAb. PVDF was stripped and analyzed with anti-total LRP6 pAb. Densitometric analysis is shown. Results represent the mean ± SD from 3 independent experiments. (*) p < 0.01 scrambled SiRNA +tPA treated cells vs scrambled SiRNA, (°) p < 0.01 SiRNA LRP1+ tPA treated cells vs SiRNA LRP1. b Evaluation of LRP1 expression after 72 h siRNA transfection by Western blot analysis using anti-LRP1 pAb. PVDF was stripped and analyzed with anti-actin mAb. A scrambled siRNA was used as control. Bar graph to the right shows densitometric analysis. Results represent the mean ± SD from three independent experiments. (*) p < 0.01 SiRNA LRP1 vs scrambled siRNA

Article Snippet: The beads were removed by centrifugation (500×g for 1 min) and the supernatants were incubated with goat anti-LRP6 Ab (R&D Systems, Minneapolis, MI, USA) at 4 °C overnight.

Techniques: Phospho-proteomics, Western Blot, Expressing, Transfection, Control

Journal: Journal of Cell Communication and Signaling

Article Title: LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells

doi: 10.1007/s12079-020-00551-w

Figure Lengend Snippet: Involvement of “lipid rafts” on LRP6 phosphorylation and β-catenin phosphorylation induced by tPA in SK-N BE2 cells. a SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with 5 mM MβCD, were analyzed by Western blot, using anti-phospho LRP-6 pAb. PVDF was stripped and analyzed with anti-total LRP6 pAb. Densitometric analysis is shown. Results represent the mean ± SD from 3 independent experiments. (*) p < 0.01 tPA treated cells vs untreated cells; (§) p < 0.01 MβCD +tPA treated cells vs tPA treated cells. b SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with 5 mM MβCD were analyzed by Western blot, using anti-phospho β-catenin pAb PVDF was stripped and analyzed with anti-β-catenin mAb. Densitometric analysis is shown. Results represent the mean ± SD from 3 independent experiments. (*) p < 0.01 tPA treated cells vs untreated cells; (§) p < 0.01 MβCD +tPA treated cells vs tPA treated cells; c SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with 5 mM MβCD were lysed in lysis buffer and subjected to cholesterol (CHOL) analysis. Neutral lipid extracts were separated by high-performance thin layer chromatography (HPTLC) using a solvent system of hexane/diethyl ether/acetic acid (70: 30: 1, v/v/v) and detected by staining with 2% copper acetate solution in 8% phosphoric acid and subsequent heating at 120 °C for 15 min. (*) p < 0.01 MβCD treated cells vs untreated cells; (°) p < 0.01 MβCD +tPA treated cells vs tPA treated cells

Article Snippet: The beads were removed by centrifugation (500×g for 1 min) and the supernatants were incubated with goat anti-LRP6 Ab (R&D Systems, Minneapolis, MI, USA) at 4 °C overnight.

Techniques: Phospho-proteomics, Western Blot, Lysis, High Performance Thin Layer Chromatography, Solvent, Staining

Journal: Nature communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin.

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: Fig. 6 | Oncogenic APC alters interactions between Wnt receptors and their effectors. To examine the effect of oncogenic APC on interactions between Wnt receptors and key lipids, cells co-expressing EGFP-tagged A, C Fzd7 or B, D LRP6 and mCherry-tagged A, B D4H or C, D pleckstrin homology (PH) domain of phos- pholipase C δ1 (PLC-δ1, PI(4,5)P2 sensor) were used to perform FLIM FRET. E To examine the effect of oncogenic APC on PI(4,5)P2 plasma membrane levels, cells expressing EGFP-tagged PLC-δ1-PH were used to measure membrane-associated EGFP fluorescence intensity using flow cytometry. EGFP plasma membrane fluor- escence intensity was normalized to total EGFP fluorescence intensity. To examine the effect of oncogenic APC on the interactions between Dvl1 and key lipids, cell co- expressing EGFP-tagged F, G Dvl1 and mCherry-tagged F D4H or G PLC-δ1 were used to perform FLIM FRET. YAMC, IMCE,and IMCE βcat cellswere pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or phenylarsine oxide (PAO) (20

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R andDSystems,Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: Expressing, Clinical Proteomics, Membrane, Cytometry

Journal: Nature communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin.

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: Fig. 7 | Oncogenic APC enhances macromolecular interactions within Wnt receptor nanoscale signaling platforms. For in vitro FLIM-FRET experiments, cells co-expressing EGFP- and mCherry-tagged A Fzd7 or B LRP6 or C EGFP-tagged LRP6 and mCherry-tagged Fzd7 were used to perform homo- and hetero-clustering FLIM-FRET analyses, respectively. To examine the effect of oncogenic APC on the interactions between Dvl1 and Wnt receptors, cells co-expressing EGFP-tagged D Fzd7 or E LRP6 and mCherry-tagged Dvl1 were used to perform FLIM-FRET. To examine the effect of oncogenic APC on plasma membrane Wnt receptor locali- zation, cells co-expressing EGFP-tagged F Fzd7 or G LRP6 and tH-RFP were used to perform FLIM-FRET analyses. For FLIM-FRET experiments, YAMC, IMCE, and IMCE

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R andDSystems,Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: In Vitro, Expressing, Clinical Proteomics, Membrane