MyD88^L265Pis secreted from the cells in EVs, which can activate signaling pathway in recipient cells. EV_L265P and EV_CONT were isolated and (A) MyD88 (protein load 10 μg) and Tsg101 or (B) IRAK4 (protein load 30 μg) and Tsg101 were detected by WB. (C) HEK293 cells expressing luciferase under NF-κB promotor and Renilla luciferase for normalization were stimulated with EV_L265P and EV_CONT (12 μg/mL) for 24 hours. EV_MWCL were isolated and (D) MyD88 (protein load 10 μg) and Tsg101 or (F) IRAK4 (protein load 30 μg) and Tsg101 were detected by WB. (E) EV_MWCL were isolated, fixed, and sections were prepared for immune labeling with anti-MyD88 antibody and protein A conjugated to 10-nm gold. Transmission electron microscopy (TEM) was performed. (G) HEK293 cells expressing luciferase under NF-κB promotor and Renilla luciferase for normalization were stimulated with EV_MWCL and EV_CONT (80 μg/mL) for 24 hours. Negative controls are transfected but unstimulated cells. Dual luciferase tests for NF-κB activity (NF-κB act.) were performed.