Anti edg 1

Human PBMCs: For the apoptosis test, anti-AnnexinV-FITC (BD Biosciences, CA, USA) was used. To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA). For CCR7 expression, we used anti-CCR7-APC (Biolegend, CA, USA).
Murine lymphocytes: The phenotype of murine lymphocytes was analyzed by flow cytometry using anti-CD3e-FITC, anti-CD19-PB, anti-CCR7-APC, anti-CD44-FITC, anti-CD62L-APC, and anti-TCR-β-PB (Biolegend, CA, USA).
To eliminate the red blood cells, RBC lysis buffer (Beit Haemek, Israel) or BD FACS lysing solution (BD Biosciences, CA, USA) was added into murine blood and splenocyte samples. Flow cytometry was performed using the MACSQuant® Analyzer (Miltenyi Biotech, Germany), and the data were analyzed using FCS Express V3 software.

The following primary antibodies and dilutions were used: rat anti-BrdU (1:300, Abcam, Cambridge, UK); rabbit monoclonal anti-EDG1 (1:1000, Abcam); goat anti-DcX,(1:150, Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-NeuN (1:200, Chemicon, Billerica, MA, USA); rabbit polyclonal anti-p-p44/42 and p44/42 MAPK (1:1000, Cell Signaling, Danvers, MA, USA).
The following reagents were used in proliferation and survival assay: fingolimod, fingolimod-phosphate (Novartis, Basel, Switzerland), Human BDNF (Novus Biologicals, Iowa City, IA, USA), Anti-BDNF (mab#9 Developmental Studies Hybridoma Bank, Iowa City, IA, USA), U0126 (Sigma-Aldrich) and PTX (Sigma-Aldrich).