Anti mr

Protein expression of MR and GPER and activity of ERK1/2 protein were determined in endothelium-intact vessels by western blot analysis. After the incubation protocols were performed, vessels were frozen in liquid nitrogen and proteins were extracted, separated (40 μg) by electrophoresis on 10% polyacrylamide gels and transferred to nitrocellulose membranes. Non-specific binding sites were blocked with 1% bovine serum albumin in Tris-buffered saline solution with Tween (0.1%) for 1 h at 24°C. Membranes were incubated with antibodies (at the indicated dilutions) overnight at 4°C. Antibodies were as follows: anti-MR (1:300, Abcam), anti-GPER (1:300, Abcam), anti-ERK1/2 (1:1000, Cell Signaling), anti-phospho (pERK)1/2 (Thr202/Tyr201; 1:1000, Cell Signaling), and anti-β-actin (1:3000, Cell Signaling). After incubation with secondary antibodies, signals were revealed by chemiluminescence, visualized by autoradiography and quantified densitometrically. Results were normalized to β-actin expression and expressed as units relative to the control.

Cellular adhesion assay was performed as previously reported [43 (link)]. Briefly, FhTeg and BSA were fluorescently labelled with PF-647 or PF-488 using the Promofluor labelling kits according to the manufacturer’s recommendations (Promokine, Heidelberg, Germany). Where indicated cells were pre-incubated with EGTA (10mM, Sigma-Aldrich), anti-MR (1 μg ml^-1, clone: 15–2, Abcam, Cambridge, UK), mannan (0.1–1 mg ml⁻¹, Sigma-Aldrich), GalNAc-4S (1-25mM, Sigma-Aldrich), for 45 min at 37°C prior to addition of fluorescently labelled FhTeg in the stated concentrations at four degrees. As control for non-specific binding, cells were incubated with fluorescently labelled BSA at four degrees. After extensive washes, binding was analysed by flow cytometry (BD FACSAria or FACSCanto, BD Biosciences), using FacsDiva (BD Biosciences) and FlowJo Software (TreeStar, Ashland, OR, USA).
For microscopy studies, after the final incubation, cells were washed with PBS and fixed in 4% paraformaldehyde. After extensive rinsing, cells were resuspended in Vectashield anti-fading solution with DAPI (Vector Laboratories), mounted on slides and viewed using a Leica DM IL LED microscope as described above.