pathscan elisa kit  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

doi: 10.1016/j.isci.2023.107548

Figure Lengend Snippet: Pi-induced activation of VEGFR2 is required for Pi-induced ERK1/2 phosphorylation and caspase-9 cleavage (A) HCs were pre-treated for 2 h with the VEGFR2 inhibitors brivanib and vandetanib followed by a 10 min treatment with 7 mM Si or 7 mM Pi. Western analyses were performed using anti-pERK1/2 and anti-tERK1/2 antibodies. (B) VEGFR2 mRNA expression day 3 (proliferative) and day 15 (hypertrophic) chondrocytes was evaluated by rt-qPCR and normalized for actin in the same sample. (C) Protein lysates from similar samples were subjected to Western analyses using anti-VEGFR2 and anti-GAPDH antibodies. (D) Densitometric quantification of western analyses was performed using blots obtained from three independent chondrocyte preparations. Data represent mean +/− the SD; p value is indicated above the line. (E) HCs were pre-treated for 2 h with brivanib or vandetanib, followed by 18 h of treatment with 7 mM Si or 7 mM Pi. Western analyses were performed using anti-caspase-9 antibody. All data are representative of that obtained from at least three independent chondrocyte preparations. (F) HCs were pre-treated for 2 h with ZM 323881 HCl (VEGFR2 inhibitor), Cl-1033 (EGFR inhibitor), or PDGFR-TKI (PDGFR Tyrosine Kinase Inhibitor IV), followed by 18 h of treatment with 7 mM Si or 7 mM Pi. Western analyses were performed using anti-caspase-9 antibody. All data are representative of that obtained from at least three independent chondrocyte preparations.

Article Snippet: Membranes were blocked with 5% non-fat dried milk (BioRad, USA) or 5% BSA (Sigma Aldrich, St. Louis, USA) prior to incubation with antibodies against pErk1/2 (1:1000; Cell Signaling Technologies), Erk1/2 (1:1000; Cell Signaling Technologies), Cleaved Caspase 9 (1:500, Cell Signaling Technologies), pVEGFR2-Tyr996 (1:1000, Cell Signaling Technologies), total VEGFR2 (1:1000, Bioss), used for evaluating VEGFR2 knockdown, and total VEGFR2 (1:1000, Cell Signaling Technologies) used for Western blot analysis.

Techniques: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

doi: 10.1016/j.isci.2023.107548

Figure Lengend Snippet: Pi and VEGF ligands induce VEGFR2 tyrosine 996 phosphorylation, ERK1/2 phosphorylation, and caspase-9 cleavage (A) HCs were pretreated with the MEK inhibitor U0126 (30 μM) for 1 h, followed by treatment with 7 mM Si, 7 mM Pi, rhVEGFA 121, or rhVEGFA 165 (50 ng/mL) for 10 min. Western analyses were performed using anti-p(Y996) VEGFR2 and anti-total VEGFR2 antibodies. (B) HCs were pretreated with the MEK inhibitor U0126 (30 μM) for 1 h, followed by treatment with 7 mM Si, 7 mM Pi, rhVEGFA 121, or rhVEGFA 165 (50 ng/mL) for 10 min. Western analyses were performed using anti-pERK1/2 and anti-tERK1/2 antibodies. (C) HCs were treated with 7 mM Si, 7 mM Pi, rhVEGFA 121 or rhVEGFA 165 for 18 h. Western analyses were performed using anti-caspase-9 antibody. Data are representative of that obtained from at least three independent chondrocyte preparations. Cl-Casp9, Cleaved caspase-9.

Article Snippet: Membranes were blocked with 5% non-fat dried milk (BioRad, USA) or 5% BSA (Sigma Aldrich, St. Louis, USA) prior to incubation with antibodies against pErk1/2 (1:1000; Cell Signaling Technologies), Erk1/2 (1:1000; Cell Signaling Technologies), Cleaved Caspase 9 (1:500, Cell Signaling Technologies), pVEGFR2-Tyr996 (1:1000, Cell Signaling Technologies), total VEGFR2 (1:1000, Bioss), used for evaluating VEGFR2 knockdown, and total VEGFR2 (1:1000, Cell Signaling Technologies) used for Western blot analysis.

Techniques: Western Blot

Journal: iScience

Article Title: Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

doi: 10.1016/j.isci.2023.107548

Figure Lengend Snippet: Chondrocyte-specific knockdown of VEGFR2 in vivo (A) rt-qPCR analysis of RNA from cultured hypertrophic chondrocytes isolated from VEGFR2 f/f and Col2Cre; VEGFR2 f/KDR mice. (B) Western analyses of cultured hypertrophic chondrocytes isolated from VEGFR2 f/f and Col2Cre; VEGFR2 f/KDR mice were performed using anti-VEGFR2 and anti-GAPDH antibodies. (C) Densitometric quantitation of western analyses was performed using blots from three independent chondrocyte preparations. Data represent the mean ± SD; of that obtained from three to four experiments; the p value is indicated above the line.

Article Snippet: Membranes were blocked with 5% non-fat dried milk (BioRad, USA) or 5% BSA (Sigma Aldrich, St. Louis, USA) prior to incubation with antibodies against pErk1/2 (1:1000; Cell Signaling Technologies), Erk1/2 (1:1000; Cell Signaling Technologies), Cleaved Caspase 9 (1:500, Cell Signaling Technologies), pVEGFR2-Tyr996 (1:1000, Cell Signaling Technologies), total VEGFR2 (1:1000, Bioss), used for evaluating VEGFR2 knockdown, and total VEGFR2 (1:1000, Cell Signaling Technologies) used for Western blot analysis.

Techniques: In Vivo, Quantitative RT-PCR, Cell Culture, Isolation, Western Blot, Quantitation Assay

Journal: iScience

Article Title: Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

doi: 10.1016/j.isci.2023.107548

Figure Lengend Snippet: Chondrocyte-specific knockdown of VEGFR2 impairs growth plate development in vivo (A–C) H&E staining was performed on humeral sections of e15.5 (5A), P0 (5B), and P8 (5C) mice. Brackets on H&E stained images at e15.5 indicate the region of vascular invasion. Brackets on H&E stained images at P0 and P8 indicate the hypertrophic chondrocyte layer. Immunohistochemistry (IHC) for Endomucin, an endothelial cell marker, demonstrated reduced vascular invasion in Col2Cre; VEGFR2 f/KDR embryos. IHC for Collagen type X (Coll-X), a marker of hypertrophic chondrocytes, demonstrated expansion of the HC layer in these embryos. A reduction in the number of pERK1/2-positive HCs in Col2Cre; VEGFR2 f/KDR embryos was observed at e15 and P0 (5A and 5B). TUNEL labeling (arrows) revealed a decrease in the number of apoptotic late HCs in Col2Cre; VEGFR2 f/KDR mice. Nuclei are stained blue with DAPI. TUNEL-positive cells were quantitated in the distal two rows of the HC layer at e15.5 (5A), P0 (5B), and P8 (5C). Data represent the mean ± SD of that obtained from four mice per group. The p value is indicated above the line. The scale bar represents 100 μM.

Article Snippet: Membranes were blocked with 5% non-fat dried milk (BioRad, USA) or 5% BSA (Sigma Aldrich, St. Louis, USA) prior to incubation with antibodies against pErk1/2 (1:1000; Cell Signaling Technologies), Erk1/2 (1:1000; Cell Signaling Technologies), Cleaved Caspase 9 (1:500, Cell Signaling Technologies), pVEGFR2-Tyr996 (1:1000, Cell Signaling Technologies), total VEGFR2 (1:1000, Bioss), used for evaluating VEGFR2 knockdown, and total VEGFR2 (1:1000, Cell Signaling Technologies) used for Western blot analysis.

Techniques: In Vivo, Staining, Immunohistochemistry, Marker, TUNEL Assay, Labeling

Journal: iScience

Article Title: Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

doi: 10.1016/j.isci.2023.107548

Figure Lengend Snippet: Dietary phosphate restriction exacerbates the growth plate phenotype of ColII-Cre+; VEGFR2 f/KDR mice H&E staining was performed on tibial sections of P24 mice weaned onto a phosphate-restricted diet on d18. Immunohistochemistry for Collagen type X (Coll-X) showed expansion of the HC layer in Col2Cre; VEGFR2 f/KDR mice. Quantification of the number of HCs per column in the growth plates is shown. TUNEL labeling (arrows) revealed a reduction in the number of apoptotic HCs in Col2Cre; VEGFR2 f/KDR mice. Nuclei are stained blue with DAPI. TUNEL-positive cells were quantitated in the distal two rows of the HC layer. Data represent the mean ± SD of that obtained from four mice per group. The p value is indicated above the line. The scale bar represents 100 μM. Chow is regular diet and LPD represents low-phosphate diet.

Article Snippet: Membranes were blocked with 5% non-fat dried milk (BioRad, USA) or 5% BSA (Sigma Aldrich, St. Louis, USA) prior to incubation with antibodies against pErk1/2 (1:1000; Cell Signaling Technologies), Erk1/2 (1:1000; Cell Signaling Technologies), Cleaved Caspase 9 (1:500, Cell Signaling Technologies), pVEGFR2-Tyr996 (1:1000, Cell Signaling Technologies), total VEGFR2 (1:1000, Bioss), used for evaluating VEGFR2 knockdown, and total VEGFR2 (1:1000, Cell Signaling Technologies) used for Western blot analysis.

Techniques: Staining, Immunohistochemistry, TUNEL Assay, Labeling

Journal: iScience

Article Title: Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

doi: 10.1016/j.isci.2023.107548

Figure Lengend Snippet:

Article Snippet: Membranes were blocked with 5% non-fat dried milk (BioRad, USA) or 5% BSA (Sigma Aldrich, St. Louis, USA) prior to incubation with antibodies against pErk1/2 (1:1000; Cell Signaling Technologies), Erk1/2 (1:1000; Cell Signaling Technologies), Cleaved Caspase 9 (1:500, Cell Signaling Technologies), pVEGFR2-Tyr996 (1:1000, Cell Signaling Technologies), total VEGFR2 (1:1000, Bioss), used for evaluating VEGFR2 knockdown, and total VEGFR2 (1:1000, Cell Signaling Technologies) used for Western blot analysis.

Techniques: Recombinant, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, In Situ, Software

Journal: BMC Biochemistry

Article Title: Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

doi: 10.1186/1471-2091-11-43

Figure Lengend Snippet: PlnDI stimulates VEGFR-2 (Tyr-951) phosphorylation . Human bone marrow endothelial cells without cell surface HS were incubated with VEGF 165 or PlnDI for 0, 2, 5, 10 and 20 minutes. Cell lysates were analyzed for VEGFR-2 phosphorylation by Western blot using anti-phospho and total VEGFR-2 tyrosine residue 951 specific antibodies. Time dependant increase in VEGFR-2 phosphorylation induced by: (A) VEGF 165 and (B) PlnDI. (C) PlnDI induced VEGFR-2 phosphorylation: Requirement for CS and HS chains. Exogenous heparin served as a positive control and GAPDH as loading control. Horizontal bars represent the densitometric scanning of bands outlined in rectangular boxes. Data are presented as the mean density values (DV) from triplicate determinations ± SEM. (*) different from 0 min or media. (δ) different from PlnDI. p < 0.05.

Article Snippet: Rabbit polyclonal antibodies for phospho- and total- VEGFR-2, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Danvers, MA), respectively.

Techniques: Incubation, Western Blot, Positive Control

Journal: BMC Biochemistry

Article Title: Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

doi: 10.1186/1471-2091-11-43

Figure Lengend Snippet: PlnDI/VEGF 165 mixtures enhance VEGFR-2 (Tyr-951) phosphorylation . Human bone marrow endothelial cells without cell surface HS were incubated with PlnDI/VEGF 165 mixtures for either 0, 2, 5, 10, or 20 min. Cell lysates were analyzed for VEGFR-2 (A and B) or Akt (C) phosphorylation by Western blot using anti-phospho and total VEGFR-2 (tyrosine residue 951) and Akt specific antibodies. (B) PlnDI/VEGF 165 enhanced VEGFR-2 phosphorylation (at min 2.5): Requirement of CS and HS chains on PlnDI. Horizontal bars represent the densitometric scanning of bands outlined in rectangular boxes. GAPDH was assessed as a loading control. Data are presented as the mean of three independent experiments ± SEM. (*) different from 0 min; (γ) different from VEGF 165 ; (φ) different from PlnDI+VEGF 165 . p < 0.05.

Article Snippet: Rabbit polyclonal antibodies for phospho- and total- VEGFR-2, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Danvers, MA), respectively.

Techniques: Incubation, Western Blot

Journal: BMC Biochemistry

Article Title: Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

doi: 10.1186/1471-2091-11-43

Figure Lengend Snippet: PlnDI binds NRP-1 and VEGFR-2 . Recombinant NRP-1 and VEGFR-2 coated plates were incubated with PlnDI +/- VEGF 165 or VEGF 121 . PlnDI binding was detected and quantified using an ELISA based approach that employed anti-PlnDI specific antibodies. For competitive inhibition assays, PlnDI was pre-incubated with heparin [1 μg/ml]. (A) PlnDI binding to VEGFR-2. (B) PlnDI binding to NRP-1. Data are presented as a percentage of total bound. (*) different from PlnDI. p < 0.05.

Article Snippet: Rabbit polyclonal antibodies for phospho- and total- VEGFR-2, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Danvers, MA), respectively.

Techniques: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: BMC Biochemistry

Article Title: Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

doi: 10.1186/1471-2091-11-43

Figure Lengend Snippet: Model: PlnDI interactions with the VEGFR-2 signaling complex . N-terminal domain I of perlecan, unbound or bound to VEGF 165 , liberated by proteolytic cleavage (scissors) during extracellular matrix turnover stimulates angiogenesis by direct interactions with: 1) NRP-1; 2) VEGFR-2; and 3) NRP-1 and VEGFR-2. PlnDI may enhance VEGF 165 stimulated pro-angiogenic events by stabilizing NRP-1/VEGF 165 /VEGFR-2 interactions.

Article Snippet: Rabbit polyclonal antibodies for phospho- and total- VEGFR-2, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Danvers, MA), respectively.

Techniques: