Journal: Cellular signalling

Article Title: Cripto-1 enhances the canonical Wnt/β-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

doi: 10.1016/j.cellsig.2012.09.024

Figure Lengend Snippet: Fig. 4. Cripto-1 binds to LRP5 and LRP6 in 293T cells. 293T cells were co-transfected with LRP5-tGFP or LRP6-GFP expression vectors together with Cripto-1 full-length or deleted expression plasmids. 3x-FLAG Cripto-1 was immunoprecipitated using an anti-FLAG mouse monoclonal antibody and the immunoprecipitated proteins were analyzed by Western blot with anti-tGFP, anti-GFP and anti-FLAG mouse monoclonal antibodies (A and C). LRP5-tGFP or LRP6-GFP was immunoprecipitated with anti-tGFP or anti-GFP mouse monoclo- nal antibody and immunoprecipitated proteins were analyzed by Western blot with anti-tGFP, anti-GFP and anti-FLAG mouse monoclonal antibodies (B and D). Cell lysates of 293T cells transiently transfected with various expression vectors as described above were analyzed by Western blot for Cripto-1 using an anti-FLAG antibody, for LRP5 using anti-tGFP antibody and for LRP6 using anti-GFP antibody (A through D).

Article Snippet: Monoclonal mouse LRP6 antibody (R&D Systems) was diluted to 20 μg/ml.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Bioprocessing

Journal: Cellular signalling

Article Title: Cripto-1 enhances the canonical Wnt/β-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

doi: 10.1016/j.cellsig.2012.09.024

Figure Lengend Snippet: Fig. 5. Cripto-1 binds to LRP5 and LRP6 co-receptors on the cell surface. 293T cells transiently transfected with Cripto-1, LRP5 and/or LRP6 expression plasmids were incubated with 2 mM NHS-PEG4-biotin and then lysed using RIPA buffer. Following immunoprecipitation with anti-FLAG or anti-GFP monoclonal antibodies, cell surface biotinylated proteins were isolated by adding streptavidin agarose beads to the immunoprecipitated proteins for 3 h at 4 °C. A and C show co-immunoprecipitation of LRP5-tGFP (A) or LRP6-GFP (C) with 3x-FLAG CR-1 WT only in the presence of biotin. B and D show co-immunoprecipitation of 3x-FLAG CR-1 WT with LRP5-tGFP (B) or LRP6-GFP (D) only in the presence of biotin. Cell lysates of immunoprecipitated samples were also analyzed by Western blot to ensure expression of transfected plasmids. (E) Endogenous Cripto-1 and LRP6 co-immunoprecipitate in NCCIT human embryonal carcinoma cells. NCCIT protein lysates were immunoprecipitated with an anti-Cripto-1 (V-17) rabbit polyclonal antibody and probed with an LRP6 monoclonal antibody (E) or immunoprecipitated with an anti-LRP6 monoclonal antibody and probed with and anti-Cripto-1 rabbit polyclonal antibody (F). * indicates the immunoglobulin light chain.

Article Snippet: Monoclonal mouse LRP6 antibody (R&D Systems) was diluted to 20 μg/ml.

Techniques: Transfection, Expressing, Incubation, Immunoprecipitation, Bioprocessing, Isolation, Western Blot

Journal: Cellular signalling

Article Title: Cripto-1 enhances the canonical Wnt/β-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

doi: 10.1016/j.cellsig.2012.09.024

Figure Lengend Snippet: Fig. 6. Cripto-1 and LRP6 co-localize on the cell membrane. 293T/Cripto-1 cells transiently transfected with LRP6-GFP expression vector were stained with anti-LRP6 (green) and anti-Cripto-1 (red) antibodies. Nuclei were visualized by Hoechst 33258 (blue). The merged image shows significant co-localization of Cripto-1 and LRP6 on the cell membrane. The arrows indicate clear examples of co-localization.

Article Snippet: Monoclonal mouse LRP6 antibody (R&D Systems) was diluted to 20 μg/ml.

Techniques: Membrane, Transfection, Expressing, Plasmid Preparation, Staining

Journal: Cellular signalling

Article Title: Cripto-1 enhances the canonical Wnt/β-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

doi: 10.1016/j.cellsig.2012.09.024

Figure Lengend Snippet: Fig. 7. β-Catenin stabilization and LRP5/6 phosphorylation in F9 and F9 Cripto-1−/−cells treated with rhWnt3a. (A) F9 and F9 Cripto-1−/−cells were stimulated with different con- centrations of rhWnt3a and activated β-catenin was immunoprecipitated with GST-E-cadherin. Glutathione agarose beads were then added to immunoprecipitated proteins and eluted proteins were analyzed by Western blot using an anti-β-catenin anti- body. Densitometric analysis of immunoprecipitated β-catenin was performed with Image J program. Protein lysates were analyzed by Western blot for β-catenin, Cripto-1 and β-actin expression (B) Western blot analysis of phosphorylated LRP5/6 in F9 and F9 Cripto-1−/−cells after rhWnt3a treatment. Density ratio (P/T) corresponds to phosphor- ylated LRP6 (P-LRP6) divided by total LRP6 (LRP6).

Article Snippet: Monoclonal mouse LRP6 antibody (R&D Systems) was diluted to 20 μg/ml.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Expressing

Journal: Cellular signalling

Article Title: Cripto-1 enhances the canonical Wnt/β-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

doi: 10.1016/j.cellsig.2012.09.024

Figure Lengend Snippet: Fig. 12. Proposed model of Cripto-1 modulation of the Wnt/β-catenin signaling path- way. Cripto-1 can regulate the Wnt/β-catenin signaling pathway through the binding of LRP5/6, which also bind to caveolin-1 in the lipid raft as well as Cripto-1. Cripto-1 facilitates the phosphorylation of LRP5/6 induced by Wnt3a. The phosphorylated LRP6 leads to recruitment of the Axin complex (Axin, APC and GSK3β) to the Wnt re- ceptor complex. The receptor complex is then internalized via a caveolin-mediated route to induce the cytoplasmic accumulation of β-catenin. β-catenin can then form a complex with Tcf/Lef and activate the transcription of target genes after translocation to the nucleus. Cripto-1 also might interfere with the internalization of LRP5/6 that is facilitated by Dkk1, which is a potent inhibitor of the Wnt/β-catenin signaling pathway.

Article Snippet: Monoclonal mouse LRP6 antibody (R&D Systems) was diluted to 20 μg/ml.

Techniques: Binding Assay, Phospho-proteomics, Translocation Assay

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

Journal: eLife

Article Title: Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice

doi: 10.7554/elife.46134

Figure Lengend Snippet: Figure 1. Design and validation of FLAgs as activators of the Wnt-bcatenin pathway. (A) Surface plasmon resonance (SPR) binding kinetics of FP Fab. Kinetics were derived from curves for soluble FP Fab interacting with immobilized FZD CRD, and ‘x’ indicates no detectable binding. (B) Anti-LRP6 Ab inhibitory activity. Inhibition of WNT1 or WNT3A signaling by indicated LRP6 Abs in the diabody-Fc format was assessed using stimulation with purified WNT3A or upon WNT1 cDNA transfection. (C) Molecular architecture of tetravalent FLAgs. (D) Activation of bcatenin signaling by FLAgs. Dose response curves are shown for the activation of a LEF/TCF reporter gene (y-axis) in HEK293T cells by serial dilutions of pan-specific FLAg proteins (FP+P- L61+1, FP+P-L63+3 and FP+P-L61+3) (x-axis). Error bars indicate SEM, n = 3. (E) Levels of bcatenin protein in RKO cells after 30 min treatment with the indicated concentrations of pan-FLAg (FP+P-L61+3). Representative blot of three replicates. (F) Time course of bcatenin and phosphorylated Disheveled- 2 (p-DVL2) protein levels in RKO cells treated with 10 nM pan-FLAg (FP+P-L61+3). Representative blot of three replicates. DOI: https://doi.org/10.7554/eLife.46134.002 The following source data and figure supplement are available for figure 1:

Article Snippet: Fc-tagged ECD fusion proteins of human (1505-LR-025) and mouse (2960-LR-025) LRP6 and mouse LRP5 (7344-LR-025/CF) were purchased from R and D Systems.

Techniques: Biomarker Discovery, SPR Assay, Binding Assay, Derivative Assay, Activity Assay, Inhibition, Purification, Transfection, Activation Assay

Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Flow cytometry sorting of the combinatorial Scl library. (A) Library expression was monitored by staining with a phycoerythrin‐conjugated antibody binding to a primary anti‐c‐Myc antibody. The purple rectangle gate indicates cells with the highest expression. (B) The library was incubated with 5 n m LRP4, and the low‐affinity library fraction was collected (purple triangle). (C) The low‐affinity library was incubated with 650 n m LRP6, and the purple rectangle represents clones that bind to LRP6.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Clone Assay

Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Identification of affinity‐reducing mutations. Heat maps demonstrating significantly enriched Scl variants in (A) LRP4 LOW library compared to Scl NAIVE library fractions; (B) LRP4 LOW LRP6 library compared to LRP4 LOW library fractions; (C) LRP4 LOW library compared to Scl NAIVE library fractions that overlap with the LRP4 LOW LRP6 library. The heat maps present the log 2 transformation of the ER (red scale bar on the right‐hand side) and highlight single mutations that significantly (A) reduce the binding affinity to LRP4, (B) reduce the binding affinity to LRP4 and retain binding to LRP6, and (C) overlap in (A) and (B). The substituting amino acids are shown on the X ‐axis, and the substituted positions are shown on the Y ‐axis. Statistical significance was determined by a two‐sided Poisson exact test and multi‐test corrected by the Benjamini–Hochberg FDR.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Transformation Assay, Binding Assay