Journal: Toxins

Article Title: P-Cresylsulfate, the Protein-Bound Uremic Toxin, Increased Endothelial Permeability Partly Mediated by Src-Induced Phosphorylation of VE-Cadherin

doi: 10.3390/toxins12020062

Figure Lengend Snippet: The effects of p-cresylsulfate (PCS) treatment on vascular endothelial (VE)-cadherin junctions and interendothelial gaps. Confluent human umbilical vein endothelial cell monolayers were treated with medium (control; A), 0.1 mM PCS (B), or 0.2 mM PCS (C) for 2 days. PCS treatment induced interendothelial gaps (indicated by arrows) visualized by immunofluorescence staining for VE-cadherin (green). The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The images are representative of three independent experiments. Scale bar: 20 µm.

Article Snippet: The cells were then washed three times with PBS and incubated for 2 h at RT with rabbit anti-VE cadherin polyclonal antibodies (1:100; sc-28644; Santa Cruz Biotechnology, Fort Worth, TX, USA) in PBS containing 0.3% Triton X-100 and 1% bovine serum albumin, followed by Alexa Fluor 488-labeled secondary antibodies (1:100; 111-545-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at RT.

Techniques: Control, Immunofluorescence, Staining

Journal: Toxins

Article Title: P-Cresylsulfate, the Protein-Bound Uremic Toxin, Increased Endothelial Permeability Partly Mediated by Src-Induced Phosphorylation of VE-Cadherin

doi: 10.3390/toxins12020062

Figure Lengend Snippet: Vascular endothelial (VE)-cadherin tyrosine phosphorylation in endothelial monolayers treated with p-cresylsulfate (PCS). Human umbilical vein endothelial cell monolayers were treated with 0.1 or 0.2 mM PCS for 2 days. The control group was treated with medium only. Equal amounts (50 µg) of protein samples were loaded in duplicate gels and separated by SDS-PAGE. The protein levels of phosphorylated-VE-cadherin (pVE-cadherin), total VE-cadherin (VE-cadherin), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected by western blotting. Following electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes and detected by either anti-pVE-cadherin or anti-VE-cadherin antibodies via immunostaining on separate membranes. The levels of GAPDH in each membrane were also detected by anti-GAPDH antibodies. (a) Representative results. Lane 1: control sample; lanes 2–3: samples treated with PCS; lane 2: 0.1 mM PCS; and lane 3: 0.2 mM PCS. (b) Protein levels (mean ± SD, n = 3 per group) of pVE relative to VE.

Article Snippet: The cells were then washed three times with PBS and incubated for 2 h at RT with rabbit anti-VE cadherin polyclonal antibodies (1:100; sc-28644; Santa Cruz Biotechnology, Fort Worth, TX, USA) in PBS containing 0.3% Triton X-100 and 1% bovine serum albumin, followed by Alexa Fluor 488-labeled secondary antibodies (1:100; 111-545-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at RT.

Techniques: Phospho-proteomics, Control, SDS Page, Western Blot, Electrophoresis, Immunostaining, Membrane

Journal: Toxins

Article Title: P-Cresylsulfate, the Protein-Bound Uremic Toxin, Increased Endothelial Permeability Partly Mediated by Src-Induced Phosphorylation of VE-Cadherin

doi: 10.3390/toxins12020062

Figure Lengend Snippet: Schematic diagram of increased endothelial permeability in human umbilical vein endothelial cell monolayers exposed to p-cresylsulfate (PCS). (A) Control endothelial cells (ECs) exhibit endothelial barrier integrity with intercellular junctions. (B) In ECs exposed to PCS, disruption of the endothelial barrier following dissociation of endothelial adherens junctions is via Src-mediated phosphorylation of VE-cadherin.

Article Snippet: The cells were then washed three times with PBS and incubated for 2 h at RT with rabbit anti-VE cadherin polyclonal antibodies (1:100; sc-28644; Santa Cruz Biotechnology, Fort Worth, TX, USA) in PBS containing 0.3% Triton X-100 and 1% bovine serum albumin, followed by Alexa Fluor 488-labeled secondary antibodies (1:100; 111-545-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at RT.

Techniques: Permeability, Control, Disruption, Phospho-proteomics