バリデーション済みの抗スフィンゴシン1リン酸1レセプター（S1PR1）／EDG1抗体 Highly validated monoclonal antibody to S1PR1 - EDG1

抗スフィンゴシン1リン酸1レセプター（S1PR1）／EDG1抗体をご紹介します。

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Immunofluorescence detection of S1P1 receptors in transfected cells and HUVEC
Endogenous S1P1 receptors were detected by confocal microscopy in HUVEC and in transfected CHO cells stably expressing S1P1- GFP with an anti-S1P1 monoclonal antibody (1/100, w/v). CHO-K1 cells were used as negative control. S1P1-GFP receptors were detected by fluorescence (excitation 488 nm, emission 500-550 nm) (a, b, i, j, green fluorescence) and immunofluorescence (Cy3-Ac II antimouse, excitation 559 nm, emission 570-625 nm) with an anti-S1P1 monoclonal antibody (c, d, k, l, red fluorescence). Nuclei were stained with TO-PRO-3 (excitation 635 nm, emission 655-755 nm, e, f, m, and n blue fluorescence). Merge images are presented (g, h, o, p). HUVEC were stained with anti-S1P1 primary antibody and Alexa-488 secondary antibody (excitation 488 nm, emission 500-550 nm). HUVEC control was realized with omitting anti-S1P1 primary antibody.

Detection of S1P1 receptors in transfected cells and human umbilical vein endothelial cells (HUVEC)
Lane 1: CHO-K1 cell membranes. Lane 2: S1P1-GFP CHO cell membranes. Lane 3: HUVEC membranes. CHO-K1 cells were stably transfected with S1P1-GFP construct (cell clone was a generous gift from Kevin R, Lynch). Samples were analysed by SDS-PAGE and immunoblotted with Mouse Anti- S1PR1 Monoclonal Antibody (clone 2B9) (1/5000 w/v dilution). S1P1 receptor was fused with the green fluorescent protein (GFP) at its C-terminus. Molecular weights: GFP: 27 kDa, S1P1: 43 kDa, S1P1-GFP: 70 kDa.

Immunoprecipitation of S1P1 receptors
S1P1-GFP receptors were immunoprecipitated with a mouse monoclonal antibody anti-S1P1 after solublization of membranes with NP40 detergent and interaction with sepharose-protein G phase. Detection of precipitated receptors was realized with a rabbit secondary anti GFP antibody coupled with HRP. Lane 1: CHO-S1P1-GFP membranes. Lane 2: Control without anti-S1P1 antibodies. Lane 3: CHO-S1P1-GFP cell membranes solubilized with NP-40. Lane 4: Immunoprecipitated sample. For control and Immunoprecipitated sample, sepharose-protein G was boiled in Laemmli buffer for 5 min at 100°C.

Comparative evaluation of different Anti-S1P1 antibodies in Flow Cytometry
S1P1 receptors were detected with anti-S1P1 antibodies (1/100) by flow cytometry in recombinant CHO cells stably expressing S1P1. GFP (red line) and in CHO-K1 cells (black line) as negative controls. S1P1-GFP receptors were detected by fluorescence (excitation 488 nm, emission 530/30 nm) (a’, b’, c’, d’) and immunofluorescence. (APC, excitation 635 nm, emission 661/16 nm) with different anti-S1P1 antibodies (a- Mab1, a-Pab1, c-Pab2 and d-# AM33115PU).