L 161 982

The MRAPs (MRAP1 and MRAP2) and 48 selected GPCRs were cloned from a wild‐type mouse cDNA library. The N‐terminal 3xHA and 2xFLAG tag were fused to GPCRs and MRAPs by PCR and the tagged cds sequence of receptor genes were all cloned into a pcDNA3.1(+) vector. Forty‐eight selected GPCRs were tagged with non‐fluorescent fragments of YFP F1 at C‐terminus. The C‐end of MRAP1 and MRAP2 were tagged with FLAG and non‐fluorescent fragments of YFP F2. Primer sequences are available on request. The validity of all constructs in this study was verified by DNA sequencing.
Prostaglandin E2, the agonist of PTGER2 and PTGER4, was purchased from TargetMol. CL‐316,243 (the agonist of ADRB3), L‐161,982 (the antagonist of PTGER4), SR 59230A (the antagonist of ADRB3), PF‐04418948 (the antagonist of Ptger2) and Antalarmin (the antagonist of CRHR1) were purchased from R&D SYSTEMS. CRF, the agonist of CRHR1, was purchased from MCE (MedChemExpress).

Intestinal microvascular endothelial cells (MIMECs) were isolated and cultured as reported previously^{4 (link),5 (link)}. When the MIMECs were cultured for 7 days, we obtained almost pure cultures of MIMECs (90% confluence). The MIMECs were subjected to the preliminary dosage selection assay, and the current dosage of TNF-α (30 ng/mL) was selected for 12 h for subsequent experiments. For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).