*4.11. ELISA*

Following siRNA knockdown, to determine the levels of molecules exported from cells, ELISAs were carried out on the media. As a control, 3 wells of nontransfected cells were treated with 50-µM MK571. For cAMP, cells were first stimulated with 100-µM forskolin for 2 h at 37 ◦C. For PGE2, cells were stimulated with 10-µg/mL lipopolysaccharides (LPS) for 24 h at 37 ◦C and with 80-nM phorbol 12-myristate 13-acetate (PMA) for 1 h at 37 ◦C, respectively. For S1P, there was no stimulation. The cell culture medium was then replaced with a fresh cell culture medium, and the cells were incubated for 18 h at 37 ◦C. The supernatants (conditioned media) were centrifuged at 1000× *g* to pellet out any floating cells and collected and stored at −80 ◦C or used immediately for determination of cAMP by using the cAMP ELISA kit (Enzo Life Sciences Inc., Exeter, UK), for the determination of PGE2, using the PGE<sup>2</sup> ELISA kit (Enzo Life Sciences Inc.), or for S1P, using the human S1P ELISA kit (Abbexa Ltd., Cambridge, UK), according to the manufacturer's instructions. The cells were also harvested for protein quantification.
