recombinant human lrp6  (Bio-Techne corporation)

Journal: Cancer letters

Article Title: Modulation of post-translational modifications in β-catenin and LRP6 inhibits Wnt signaling pathway in pancreatic cancer

doi: 10.1016/j.canlet.2016.11.026

Figure Lengend Snippet: Effect of inhibition of LRP6 and FZD1 on β-catenin-TCF signaling. (A) Transcriptional changes in β-catenin after treating MIA PaCa-2 and S2-VP10 cell lines with SiLRP6, SiFZD1 and SiLRP6+SiFZD1 for 48 h and overexpression of recombinant rLRP6 and rFZD1 plasmids for 24 h. (B) MIA PaCa-2 and S2-VP10 cytoplasmic and nuclear fractions were isolated using cytoplasmic and nuclear extraction kit. Different fractions of MIA PaCa and S2-VP10 cells were immunoblotted and probed with β-catenin antibody. (C) MIA PaCa-2 and S2-VP10 cells were transfected with TCF/LEF reporter plasmid for 18 h and treated with SiLRP6, SiFZD1 and SiLRP6+SiFZD1 for 48 h, inhibition of LRP6 and FZD were recovered using rLRP6 and rFZD1 plasmids for 24 h, luciferase activity was counted with Promega kit. (D) Expression of Cyclin D1 and c-Myc by q-RT PCR upon LRP6 and FZD silencing in MIA PaCa-2 and S2-VP10 (Supplementary Fig. 4) cell line. (E) Viability of S2-VP10 cells were measured with CCK8 after 48 h and 72 h treatment of SiLRP6, SiFZD1 and SiLRP6+SiFZD1 and after that overexpression of recombinant rLRP6 and rFZD1 plasmids for 24 h. (F) Viability of MIA PaCa-2 by CCK8. Experiment was repeated three times with similar results obtained.

Article Snippet: A rescue experiment for silencing was done by transiently transfecting pancreatic cancer lines with Recombinant (r) human LRP6 protein (R and D systems), rFZD (R and D systems) and rOGT.

Techniques: Inhibition, Over Expression, Recombinant, Isolation, Extraction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer letters

Article Title: Modulation of post-translational modifications in β-catenin and LRP6 inhibits Wnt signaling pathway in pancreatic cancer

doi: 10.1016/j.canlet.2016.11.026

Figure Lengend Snippet: Effect of Minnelide and triptolide on β-catenin/Wnt signaling. (A) Effect of Minnelide on total LRP6 and phosphor (p) LRP6 protein levels during the tumor progression. (B) Protein levels of DSH, p-β-catenin, β-TrCP were compared at different time points of triptolide treatment in MIA PaCa-2 and S2-VP10 cell lines. (C) MIA PaCa-2 cells were treated with 100 nM triptolide for (12, 24 and 48 h) immunoprecipitated with GSK-3β and immunoblotted with GSK-3β, Axin2, APC, β-TrCP and p-β-catenin antibodies, respectively. (D) MIA PaCa-2 cells were treated with 10 μM MG-132 followed by the treatment of 100 nM triptolide for 48 h and immunoblotted with β-catenin, c-Myc and Cyclin D1. The experiment was repeated two times with similar results obtained.

Article Snippet: A rescue experiment for silencing was done by transiently transfecting pancreatic cancer lines with Recombinant (r) human LRP6 protein (R and D systems), rFZD (R and D systems) and rOGT.

Techniques: Immunoprecipitation

Journal: Translational Psychiatry

Article Title: A role for APP in Wnt signalling links synapse loss with β-amyloid production

doi: 10.1038/s41398-018-0231-6

Figure Lengend Snippet: a APP overexpression enhanced Wnt-β-catenin signalling as measured by TCF/LEF transcriptional activity using SuperTOPflash luciferase reporter assay in HEK293A cells; activity was measured in relative light units (RLU). The enhancement of Wnt-β-catenin signalling was only detectable in cells also overexpressing Wnt3a (a “canonical Wnt”) and not in the presence of the “non-canonical Wnt”, Wnt5a. b APP overexpression also enhanced Wnt-PCP signalling as measured by AP1 transcriptional activity (APRE-luciferase) in HEK239A cells. The enhancement of Wnt-PCP signalling was only detectable in cells also overexpressing Wnt5a and not in the presence of Wnt3a. c APP co-immunoprecipitated from lysates of primary rat cortical neurons with Wnt-β-catenin co-receptor protein, LRP6 and with Wnt-PCP co-receptor protein, Vangl2. (The full blots are presented in the supplemental data.) d Schematic illustrating the sites of APP-LRP6 interaction determined by peptide array hybridisation. Overlay of APP peptides with recombinant extracellular domain (ECD) of LRP6 demonstrated binding between the LRP6 and APP ECDs. e Schematic illustrating the site of APP-Vangl2 interaction as elucidated by peptide array. Overlay of APP peptides with HEK293A lysates overexpressing Vangl2-HA demonstrated binding of Vangl2 to a single site in the APP-ICD. f Schematic of the precise binding regions of LRP6/Vangl2 binding within the APP sequence. Binding of LRP6 is restricted to the ECD with one region lying close to the site affected by the Swedish APP mutation. In contrast, Vangl2 binding at the APP-ICD occurs at the YENPTY motif, which has been shown to control APP internalisation. g Overexpression of LRP6 enhanced the activity of APP in Wnt-β-catenin signalling as measured by TCF/LEF transcriptional activity using SuperTOPflash luciferase reporter assay in HEK293A cells. h Overexpression of Vangl2 enhanced the activity of APP in Wnt-PCP signalling as measured by APRE luciferase reporter assay in HEK293A cells. Data are plotted as mean+/− standard deviation ( n ≥ 3). Statistical significance was determined by one-way ANOVA and post-hoc Tukey’s test (* p < 0.05; ** p < 0.01); p values are indicated for key comparisons only

Article Snippet: Arrays were incubated with recombinant human LRP6 (Fc-chimera, R&D systems) or total lysate from HEK293A cells overexpressing HA-tagged Vangl2.

Techniques: Over Expression, Activity Assay, Luciferase, Reporter Assay, Immunoprecipitation, Peptide Microarray, Hybridization, Recombinant, Binding Assay, Sequencing, Mutagenesis, Control, Standard Deviation

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

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: Recombinant human LRP6 with an Fc-tag, human ADAM8 and ADAM12, VEcadherin, and mouse LRP6 and LRP5 having a His-tag were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Activation Assay, Disruption