Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the phosphorylation of LRP6 in cardiac side population cells (CSPs) by UT/JNK signaling during mechanical stretch. ( A ) Immunofluorescent images revealed that positive signal (green) could be observed in cultured CSPs with antibody to p-LRP6 (1:200); bar: 20 μm. ( B ) Quantitative evaluation of p-LRP6 by analysis of fluorescence intensity. ( C ) P-LRP6 was detected in cultured CSPs by nanofluidic proteomic immunoassay (NIA). Peaks on the traces that represent phosphorylated isoforms of LRP6 are indicated. ( D ) NIA pseudoblot representation of p-LRP6 and bar graph of p-LRP6/GAPDH in cultured CSPs by NIA quantification. Cultured CSPs were pre-treated with PBS, urantide (Ura, 1 μM) or SP600125 (SP6, 5 μM), respectively, for 30 min, then subjected to mechanical stretch (MS) and incubated with PBS or UII (0.1 μM) for 3 hrs. Values are expressed as mean ± SEM. ** P < 0.01 versus control; ## P < 0.01 versus control; # P < 0.05 versus MS; & P < 0.05 versus MS plus UII; && P < 0.01 versus MS plus UII. The experiment was repeated for at least three times. ( E ) P-LRP6 was detected in CSPs isolated from transverse aorta constriction (TAC) or Sham mice by NIA. ( F ) NIA pseudoblot representation of p-LRP6 and bar graph of p-LRP6/GAPDH in isolated CSPs by NIA quantification. Values are expressed as mean ± SEM. Sham: n = 9; Sham+Ura: n = 6; TAC: n = 6; TAC+Ura: n = 7. Ura: urantide. ** P < 0.01 versus Sham mice; ## P < 0.01 versus TAC mice.

Article Snippet: After washing and blocking with PBS containing 2% bovine serum albumin (Sigma-Aldrich), cells were incubated with anti-phospho-LRP6 (Ser 1490) (1:100, Bioss Inc., Woburn, MA, USA) in 4°C overnight.

Techniques: Cell Culture, Fluorescence, Incubation, Isolation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the phosphorylation of LRP6 in UT cell line by activating JNK during mechanical stretch. Western blot analysis for p-LRP6 and p-JNK levels. UT cells were pre-treated with Urantide (Ura, 1 μM) for 30 min., then subjected to MS and treated with UII for 30 min. and 3 hrs. ( A ) Representative picture of p-LRP6 and p-JNK level at 30 min. after UII treatment during MS. Quantitative evaluation of p-JNK ( B ) and p-LRP6 ( C ) by analysis of Western blot. ( D ) Representative picture of p-LRP6 and p-JNK level at 3 hrs after UII treatment during MS. Quantitative evaluation of p-JNK ( E ) and p-LRP6 ( F ) by analysis of Western blot. P-JNK and p-LRP6 were analysed in UT cells pre-treated with SP600125 (SP6, 5 μM; G ) or overexpressed LRP6 ( H ) with or without UII (0.1 μM) during MS. UT cells were pre-treated with SP600125 or DMSO for 30 min., then subjected to MS and treated with UII for 3 hrs. UT cells were overexpressed with LRP6-pCS2 or pCS2 for 48 hrs, then subjected to MS and treated with UII for 3 hrs. Values are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 versus control. # P < 0.05, ## P < 0.01 versus MS. & P < 0.05, && P < 0.01 versus MS plus UII. The experiment was repeated for at least three times.

Article Snippet: After washing and blocking with PBS containing 2% bovine serum albumin (Sigma-Aldrich), cells were incubated with anti-phospho-LRP6 (Ser 1490) (1:100, Bioss Inc., Woburn, MA, USA) in 4°C overnight.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the proliferation of cardiac side population cells (CSPs) by c-Jun N -terminal kinase (JNK)-LRP6 signalling during mechanical stretch. CSPs proliferation was determined by Cell Viability Assay. Cultured CSPs were overexpressed LRP6 ( A ) or pretreated with SP600125 (SP6, 5 μM; B ) with or without UII (0.1 μM) during mechanical stretch (MS). CSPs were pre-treated with SP600125 or DMSO for 30 min., then subjected to MS and treated with UII for 48 hrs. CSPs were overexpressed with LRP6-pCS2 or pCS2 for 48 hrs, then subjected to MS and treated with UII for 48 hrs. Values are expressed as mean ± SEM. ** P < 0.01 versus MS. # P < 0.05 versus MS plus UII. The experiment was repeated for at least three times. ( C ) The ratio of CSPs per mouse was analysed by fluorescence-activated cell sorting (FACS). Representative photographs are shown. ( D ) The ratio of CSPs per mouse was calculated by FACS. SP600125 (SP6) or DMSO were injected intraperitoneally either with 40 mg/kg bodyweight every 3 days for 2 weeks. At 4 weeks after transverse aorta constriction (TAC) or sham operation, the number of CSPs was determined. Values are expressed as mean ± SEM. Sham: n = 5; Sham+SP6: n = 6; TAC: n = 6; TAC+SP6: n = 6. Ura: urantide. * P < 0.05 versus sham mice; # P < 0.05 versus TAC mice.

Article Snippet: After washing and blocking with PBS containing 2% bovine serum albumin (Sigma-Aldrich), cells were incubated with anti-phospho-LRP6 (Ser 1490) (1:100, Bioss Inc., Woburn, MA, USA) in 4°C overnight.

Techniques: Viability Assay, Cell Culture, Fluorescence, FACS, Injection

Journal: Developmental cell

Article Title: APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

doi: 10.1016/j.devcel.2018.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-LRP6 (C47E12) , Cell Signaling , Cat# 3395; RRID: AB_1950408.

Techniques: Control, Virus, Recombinant, Luciferase, Viability Assay, Reverse Transcription, Plasmid Preparation, Software

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: 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 phospholipase C δ1 (PLC-δ1, PI(4,5)P 2 sensor) were used to perform FLIM FRET. E To examine the effect of oncogenic APC on PI(4,5)P 2 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 fluorescence 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 cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or phenylarsine oxide (PAO) (20 µM, 30 min) and washed, as indicated. Subsequently, cells were incubated with Wnt3a-conditioned media or control media without Wnt3a for 30 min, washed, fixed, and imaged. For FLIM-FRET experiments, the apparent FRET efficiency was calculated from FLIM data averaged per FOV (mean ± SD, n = 10–15 FOVs containing 3–8 cells were examined per condition, exact n value is shown in each graph). For flow cytometry IL PI(4,5)P 2 experiments, cells were imaged to calculate EGFP fluorescence intensity (mean ± SEM, from n = 3 independent biological replicates, total number of cells analyzed is provided below each bar). For all experiments, statistical significance was determined by two-way ANOVA and post Tukey’s multiple comparison test. Different letters indicate significant differences between WT APC (control) and mutant APC/treatment groups (experimental) ( P < 0.05). Source data are provided as a Source data file.

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 and D Systems, 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, Fluorescence, Flow Cytometry, Incubation, Control, Comparison, Mutagenesis

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: 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 localization, 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 βcat cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or phenylarsine oxide (PAO) (20 µM, 30 min) and washed, as indicated. Subsequently, cells were incubated with Wnt3a-conditioned media or control media without Wnt3a for 30 min, washed, fixed, and imaged. The apparent FRET efficiency was calculated from FLIM data averaged per FOV (mean ± SD, from n = 10–15 FOVs containing 3–5 cells each were examined per condition, exact n value is shown in each graph). For all experiments, statistical significance was determined by two-way ANOVA and post Tukey’s multiple comparison test. Different letters indicate significant differences between WT APC (control) and mutant APC/treatment groups (experimental) ( P < 0.05). Source data are provided as a Source data file.

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 and D Systems, 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, Incubation, Control, Comparison, Mutagenesis

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: For in vivo STORM imaging experiments, isolated single colonocytes from PDOs were fixed and labeled with primary monoclonal rat Fzd7 or mouse LRP6 antibody fluorescently labeled with Alexa Fluor 647. A Model of the formation of Wnt proteolipid condensates in ordered plasma membrane nanodomains examined via STORM imaging. B Representative bright field and Voronoi images of isolated single colonocytes from CRC-PDOs labeled with primary anti-LRP6-AF647. Quantitative analysis of Fzd7 and LRP6 C , E cluster area, D , F cluster area relative frequency, G , H total number of receptor molecules inside clusters, I , J receptor molecule absolute density, K , L percentage of receptor molecules forming part of clustered regions, M , N total number of receptor clusters, and O , P cellular receptor cluster density in isolated single colonocytes from PDOs, respectively. Cluster area was calculated from STORM data averaged per region of interest (ROI) and the respective relative frequency was calculated from individual cluster distribution data (mean ± SD, from n = 50–2274 ROIs, exact n value is shown below each bar). Data associated with the number of single receptor molecules, receptor clusters, and their density was calculated from raw fluorescence intensity images converted to text (.txt) x–y coordinate files using Clus-Doc (mean ± SD, from n = 22–66 ROIs, exact n value is shown below each bar). Different letters indicate significant differences between WT APC (control) and mutant APC groups (experimental) ( P < 0.05). Source data are provided as a Source data file.

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 and D Systems, 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 Vivo, Imaging, Isolation, Labeling, Clinical Proteomics, Membrane, Fluorescence, Control, Mutagenesis

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: A Drosophila midgut-hindgut intestinal tissue model. ISCs/progenitor cells express humanized hFzd7 or hLRP6 under control of the UAS and esg-Gal4 TS . B Filipin III-stained midgut from Drosophila fed various cholesterol diets (red arrow, intestinal lumen). C Quantification of total cholesterol from Drosophila midgut. Cholesterol was calculated from luciferase luminescence data using the Amplex™ Red cholesterol assay and normalized to total protein (mean ± SD, from n = 3 independent biological replicates). D Filipin III fluorescence distribution of Drosophila intestinal epithelium (red arrow, ISCs). Effects of cholesterol on Wnt receptor organization. Flies co-expressing EGFP- and mCherry-tagged E LRP6 or F Fzd7 or G EGFP-LRP6 and mCherry-Fzd7 were used to perform FLIM-FRET in flies fed various cholesterol diets. FRET efficiency was calculated from FLIM data (mean ± SD, from n = 5–10 guts, ROIs analyzed provided below each bar, n value is shown in each graph). H Quantitative analysis of free cholesterol-induced βcat activation. 3T3 LL cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), and MβCD-cholesterol (cholesterol) (10 mM, 30 min), and incubated with control or Wnt3a-conditioned media for 24 h. Luciferase luminescence was measured using a Luciferase Assay System kit. Luciferase luminescence fold change was normalized to total protein (mean ± SD, from n = 3 independent biological replicates). I Quantitative analysis of Wnt signaling activation. The percentage of TCF-LacZ+ cells is shown. Error bars represent n = 5 independent biological replicates (mean ± SD, ~100 cells analyzed per group). J Qualitative analysis of cholesterol-induced Wnt activation. TCF-LacZ+ cells activity in Drosophila posterior midguts from control ( w 1118 ; esgGal4 TS , UAS-GFP; TCF-LacZ) and hLRP6-expressing ISCs ( w 1118 ; esgGal4 TS , UAS-GFP, UAS-hLRP6; TCF-LacZ). Flies were feed a cholesterol free, standard (Std. Diet) or high cholesterol diet for 5 days. ISCs, GFP; nuclei, DAPI. Statistical significance determined by C two-way ANOVA or E – I one-way ANOVA and post hoc Tukey’s test. Different letters indicate significant differences between treatment groups ( P < 0.05). Representative images and scale bars are provided for microscopy data. Enterocytes, ECs; visceral muscle cells, muscle; intestinal stem cells, ISCs; enteroblasts, EB. Source data file provided.

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 and D Systems, 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: Control, Staining, Luciferase, Amplex Red Cholesterol Assay, Fluorescence, Expressing, Activation Assay, Incubation, Activity Assay, Microscopy

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 2. VAP1 cleaves LRP6 at the activation region. (A) HeLa cells were incubated with or without 0.4 lgmL1 VAP1 in serum-free medium for 2 h at 37 °C. The cells (1.5 9 107 cells) were harvested with a lysis buffer (25 mM Tris/HCl, pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF, 4 mM EDTA). Samples were immunoprecipitated with an anti-LRP6 antibody. The precipitates were subjected to SDS/PAGE (10% separating gel) and western blotting by using an anti-LRP6 antibody as a primary antibody. (B) Ectodomain recombinants of human LRP6 and mouse LRP5 at 17 lgmL1 and 50 lgmL1 purified bovine fibrinogen were incubated with 0.03 lgmL1 VAP1 in PBS for 0, 1 and 3 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Human fibronectin at 15 lgmL1 was incubated with each dose of VAP1 in PBS for 1 h at 37 °C. Samples (12 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP6. Fragment sequences of 140-kDa and 60-kDa bands of LRP6 detected by mass spectroscopic analysis are shown by a double underline and dashed underline, respectively. (E) A mouse ectodomain recombinant of LRP6 at 10 lgmL1 was incubated with 0.1 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining.

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Activation Assay, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Silver Staining, Recombinant

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 3. VAP1 cleaves LRP5 at the same site as that of LRP6. (A,B) A mouse ectodomain recombinant of LRP5 at 26 lgmL1 was incubated with each dose of VAP1 (A) and VAP2 (B) in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) Mouse LRP5 ectodomain recombinant at 70 lgmL1 was incubated with 7 lgmL1 VAP1 in PBS for 1 h at 37 °C. Samples (60 lL) were subjected to SDS/PAGE (5–20% precast SDS/PAGE gel) and stained with Coomassie Brilliant Blue R250. (D) The VAP1-cleavage point is shown by a black arrow in the schematic representation of LRP5. The fragment sequences of the 140-kDa band of LRP5 detected by mass spectroscopic analysis is shown by a double underline. (E) Amino acid residues from cleavage site position P5 to P50 of LRP6 and LRP5 are aligned. The VAP1-cleavage point is shown by a black arrow.

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Recombinant, Incubation, SDS Page, Silver Staining, Staining

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 4. Substrates and cleavage points of VAP1. (A) Substrates cleaved by VAP1 are indicated. The cleaved fragments were subjected to N-terminal sequence analysis. P5 to P50 amino acid residues of the cleavage points are shown. (B) Docking model of VAP1 and the cleavage site moiety of LRP6. The inset indicates the substrate-binding cleft. VAP1 is shown by a white space-filling model. The cleavage site moiety of LRP6 is shown by a red space- filling model (in the upper figure) and by a ball-and-stick model (in the lower figure). Zn2+ and Glu336A, both of which are involved in catalysis, and Leu363A are shown by grey spheres. S10 and S30

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Sequencing, Binding Assay

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 5. LRP6 cleavage by VAP1 is involved in disruption of cell–cell junctions and haemorrhage. (A,B) A human ectodomain recombinant of LRP6 at 30 lgmL1 was incubated with 30 ngmL1 VAP1 and with 2 mgmL1 LRP-cleavage site antibody or control antibody in PBS for 1 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE and silver staining (A). Cleavage inhibition is shown by the 140-kDa fragment density score (B). The data (n = 5; error bars correspond to standard errors) were compared by Student’s t test. (C) HUVECs were incubated with 140 ngmL1 VAP1 and with 1.35 mgmL1 LRP6-cleavage site antibody or control antibody in a medium for 1 h at 37 °C. After being fixed, the cells were stained with anti-VE-cadherin antibody. Arrows indicate the remaining membrane VE-cadherin. Scale bar, 50 lm. (D,E) VAP1 with LRP6 cleavage site antiserum or control serum was intradermally injected into mice bisymmetrically. One hour later, haemorrhagic plaques on the inner surface of the skin were observed (D). Scale bar, 10 mm. Densitometry scores of VAP1-induced haemorrhage with the antiserum or control serum in each individual were compared by paired t test (E).

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Disruption, Recombinant, Incubation, Control, SDS Page, Silver Staining, Inhibition, Staining, Membrane, Injection

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 6. VAP1-cleavage site and/or LDLa domains of LRP5/6 are deleted in many venom-resitent animals. Deleted regions of LRP6 (A) and LRP5 (B) in animals are shown in schematic representation. LRP5/6 of animals associated with tolerance to snake bite-induced haemorrhage are indicated by red boxes. LRP5/6 of animals with moderate resistance and animals with unknown sensitivity to snake bite- induced haemorrhage are shown by orange and green boxes, respectively. The presented data are from NCBI RefSeq (Table S1). Except for humans, mice and rats, the data are all predicted sequences from the genome of each animal. Amino acid sequences of the cleavage site and LDLa region are shown in Fig. 7. Regarding the predicted king cobra LRP6 (ETE69657.1), although the genome sequence of the full- length mRNA region of LRP6 contains several gaps, the indicated deleted region does not contain gaps.

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Combined Bisulfite Restriction Analysis Assay, Sequencing

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 8. Recombinant human ADAM8 and ADAM12 cleave LRP6 at the same site as that of VAP1. (A) Recombinant human LRP6 at 64 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM8 for 3 h at 37 °C. Samples (10 lL) were subjected to SDS/PAGE (12% separating gel) and silver staining. (B) LRP6 at 20 lgmL1 was incubated with 20 lgmL1 recombinant human ADAM12 for 16 h at 37 °C. Samples (15 lL) were subjected to SDS/PAGE (10% separating gel) and silver staining. (C) LRP6 at 20 lgmL1 was incubated with 200 lgmL1 recombinant human ADAM12 for 18 h at 37 °C. Fragments containing C-terminal Fc peptide (20 lL) were purified with protein A–Sepharose. Samples were subjected to SDS/PAGE (15% separating gel) and silver staining. (D) The LRP6 140-kDa fragments treated with ADAM8 (A) and ADAM12 (B) and the LRP6 60-kDa fragment treated with ADAM12 (C) were analysed by mass spectrometry. Detected fragments by mass spectrometry and the proposed cleaved sites are shown.

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Recombinant, Incubation, SDS Page, Silver Staining, Mass Spectrometry

Journal: The FEBS journal

Article Title: Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

doi: 10.1111/febs.14066

Figure Lengend Snippet: Fig. 9. Hypothetical roles of ADAM and LRP5/6 in haemorrhage and invasion. (1) Cell–cell junctions with cadherin and catenin are normally stable. LRP5/6 may form a complex with cadherin and catenin. (2) Haemorrhagic SVMP, which is an ADAM-type toxin, can cleave LRP5/6 at the activation region. Cleaved LRP5/6 may form a dimer or multimer. (3) Cleaved LRP6 is involved in c-catenin and VE-cadherin relocation and in disruption of cell–cell adhesions. (4) Cleaved LRP6 mediates haemorrhage. Some animals with snake venom tolerance have deleted LRP5/6. (20) Invasion-associated ADAMs are located at the tips of invadopodia in invasive cells such as leukocytes and cancer cells. The ADAMs can cleave the same site of LRP6. (40) Cleaved LRP6 has the potential to cause disruption of cell–cell adhesions and to induce invasion. Summarizing the above, it is thought that ADAMs, as cell barrier openers, cleave novel ADAM receptors, LRP5/6, to induce haemorrhage and potentially invasion.

Article Snippet: After blocking with 5% skim milk in Tris-buffered saline, 0.1% Tween 20 buffer, the membranes were incubated with the indicated dose of antiVE-cadherin monoclonal mouse antibody clone TEA1/31 (Beckman Coulter Inc., Brea, CA, USA) or anti-human LRP6 polyclonal goat antibody (R&D Systems, Inc.).

Techniques: Activation Assay, Disruption