L alpha phosphatidylcholine palmitoyl

Cells were incubated in RPMI 1640 medium/20 mM HEPES. CXCL12 or SEMA3F were then added to the cell suspension. At each indicated time point (15 s to 2 min), an aliquot was taken from the cell suspension and mixed with the labeling buffer, consisting in 10⁻⁷M FITC-phalloidin (Sigma-Aldrich), 0.125 mg/ml L-alpha-phosphatidylcholine palmitoyl (Sigma-Aldrich), and 4.5% PFA in PBS. Staining was analyzed by flow cytometry. Mean fluorescence intensity (MFI) values obtained before addition of ligand were arbitrarily set at 100%.

Sphingosine-1-phosphate, W146, (4R)-2-Undecyl-4-thiazolidinecarboxylic acid (BML-241), FITC-phalloidin, L-alpha-phosphatidylcholine palmitoyl and fatty acid free bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St Louis, USA). Rabbit anti-phospho-Akt (catalog number: 9271), rabbit anti-Akt (catalog number: 4685), rabbit anti-p44/42 MAPK (catalog number: 4695), rabbit anti-phospho-p44/42 MAPK (catalog number: 9101) and goat anti-rabbit HRP-linked (catalog number: 7071) polyclonal or monoclonal antibodies were purchased from Cell Signaling (Danvers, USA), whereas the mouse anti-actin monoclonal antibody (clone C4—catalog number: MAB1501) was from Merck Milipore (Darmstadt, Germany) and the goat anti-mouse HRP-linked antibody (catalog number: 1031–05) was from Southern Biotech (Birmingham, USA).