Rabbit anti actin

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.

Wild‐type human full‐length TLK1 mammalian expression plasmid was purchased from Addgene (Watertown, MA, USA, cat# 98378). Wild‐type full‐length human TLK1B bacterial expression plasmid was obtained from S. Kirubakaran, Discipline of Biological Engineering, Indian Institute of Technology, Gandhinagar, India [40 (link)]. Dominant negative human TLK1 kinase‐dead mammalian expression vector was generated as previously described [41 (link)]. pEGFP‐MK5‐C1 mammalian expression vector was a kind gift from O. Morten Seternes (Department of Pharmacology,1 Institute of Medical Biology, University of Tromsø, Tromsø, Norway) [42 (link)]. Human full‐length MK5 bacterial expression plasmid was purchased from Vector Builder (Chicago, IL, USA). The following primary antibodies were used in this study: rabbit anti‐TLK1 (Thermo Fisher, Waltham, MA, USA, cat# 720397), rabbit anti‐TLK1B (laboratory‐generated), rabbit anti‐MK5 (Cell Signaling Technology, CST, Danvers, MA, USA, cat# 7419S), mouse anti‐PRAK (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, sc‐46667), rabbit anti‐phospho‐MK5 S354 (Thermo Fisher, cat# PA5‐105676), mouse anti‐GFP (Thermo Fisher, cat# MA5‐15256), rabbit anti‐GAPDH (CST, cat# 2118S) and rabbit anti‐actin (Abcam, Cambridge, MA, USA, cat# ab1801).

20 µg cell lysates were subjected to SDS–PAGE and electrically blotted on to PVDF membrane. After blocking with 2% (w/v) BSA, the blot was probed with rabbit anti-β-tubulin (Abcam), rabbit anti-actin (Abcam), rabbit anti-NMIIA (Abcam) or rabbit anti-S100P (R&D Systems). After extensive washing, anti-rabbit-HRP (Santa Cruz) 1/10,000 was used and the bands were visualised using ECL and their densities were measured using Image Lab software.