Total membranes were prepared to determine the expression levels of S1PRs. Colon samples were homogenized on ice for 3 minutes in 20 mM Tris-HCl, pH 7.5 containing 2 mM MgCl2, 0.25 M sucrose, and 1x protease inhibitors (Sigma Aldrich, Munich, Germany). Homogenates were centrifuged at 3000 x g for 10 minutes at 4°C and supernatants were subjected to another centrifugation step at 100,000 x g for 1 hour at 4°C. The pellet was suspended in 50 mM Tris-HCl, pH 7.5 containing 1x protease inhibitors cocktail (Sigma Aldrich, Munich, Germany) and 0.1% sodium dodecyle sulfate. Protein concentration was measured using the BCA-protein determination kit (Themoscientific, Ottawa, Ontario, CA) and samples were stored at -80°C for further analysis.
After boiling, samples from control and colitic colon segments (50 μg protein) were subjected to SDS-PAGE as previously described [44 (link)]. The membranes were immunoblotted with rabbit anti-S1PR1 (Cayman Chemical, MI, USA), rabbit anti-S1PR2 (Sigma Aldrich, Munich, Germany), rabbit anti-S1PR3 (Cayman Chemicals, MI, USA) or rabbit anti-actin (Abcam, MA, USA). HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch, PA, USA) was used as secondary antibody. Bands were detected using Super Signal Western Pico chemiluminescence Substrate (Thermoscientific, Ottawa, Ontario, CA) and quantified using Image J software.
After boiling, samples from control and colitic colon segments (50 μg protein) were subjected to SDS-PAGE as previously described [44 (link)]. The membranes were immunoblotted with rabbit anti-S1PR1 (Cayman Chemical, MI, USA), rabbit anti-S1PR2 (Sigma Aldrich, Munich, Germany), rabbit anti-S1PR3 (Cayman Chemicals, MI, USA) or rabbit anti-actin (Abcam, MA, USA). HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch, PA, USA) was used as secondary antibody. Bands were detected using Super Signal Western Pico chemiluminescence Substrate (Thermoscientific, Ottawa, Ontario, CA) and quantified using Image J software.