Pannexin-1 is present in a subpopulation of bovine milk-derived small extracellular vesicles.

Pannexin-1 (Panx1) is a large-pore membrane channel protein implicated in diverse physiological and pathological processes, yet its presence in extracellular vesicles has not been fully characterized. In this study, we investigated small extracellular vesicles derived from bovine milk to determine whether they contain Panx1. Using multiple complementary approaches, we show that Panx1 is present in a distinct subpopulation of milk-derived vesicles. Panx1 immunoreactivity was detected by Western blotting with antibodies targeting both the C-terminal and extracellular loop regions, and single-molecule localization microscopy revealed that a subpopulation of CD9-positive mEVs expresses Panx1. Consistently, flow cytometry-based analysis indicated that approximately half of the vesicle population was Panx1 positive. Together, these findings provide the first validated evidence that Panx1 is incorporated into bovine milk-derived small extracellular vesicles, expanding the repertoire of membrane channel proteins associated with extracellular vesicles and establishing a foundation for future studies on the functional role of Panx1 in vesicle biology and vesicle-mediated signaling.