Paracrine activation of IKKε and NF-κB by kGPCR. (A) Whole-cell lysates of HUVECs were analyzed for RelA S468p with indicated antibodies by immunoblot. (B) HUVECs were stimulated with conditioned medium (CM) of HUVEC/Vec or HUVEC/kGPCR cells for 24 h. Total RNA was extracted and analyzed by qRT-PCR. (C and D) Human ECV endothelial cells, infected with control (Vec) or IKKεK38A-expressing lentivirus (C), or treated with amlexanox (15 μM) (D), were stimulated with HUVEC/kGPCR-conditioned medium and analyzed by immunoblot for RelA. (E) HUVECs were treated with conditioned medium of HUVEC/Vec (H-Vec) or HUVEC/kGPCR (H-kGPCR), without or with amlexanox, at indicated concentrations for 18 h. Total RNA was extracted and analyzed by qRT-PCR.