Internalization of the intact EVs is necessary for signaling. EV_L265P were isolated either from the NEMO^KO cells or NEMO^wt cells. (A) HEK293 cells expressing luciferase under the NF-κB promotor and Renilla luciferase for normalization were stimulated with EV_L265P (12 μg/mL) for 24 hours. EV_L265P were isolated from HEK293 cells and (B) IRAK4^wt and IRAK4^KO BMDMs were stimulated for 16 hours (30 μg/mL). LPS was a positive control (10 ng/mL). RNA was isolated and qPCRs for Rantes was performed. (C) EV_L265P and EV_CONT were submitted to 3 frozen/thaw cycles and then added to cells (12 μg/mL). (D-F) Dynasore (30 and 50 μM), LY294002 (50 μM), or ibrutinib (4 μM) were added to cells 1 hour prior stimulation with EV_L265P and EV_CONT (12 μg/mL) or EV_MWCL (80 μg/mL) and EV_CONT (80 μg/mL). HEK293 cells expressing luciferase under NF-κB promotor and Renilla luciferase for normalization were stimulated for 24 hours. Negative controls are transfected but unstimulated cells. Dual luciferase tests for NF-κB activity were performed.