In our studies on the unconventional secretion mechanism of FGF2, we identified three key lipid species-cholesterol, sphingomyelin (SM), and PI(4,5)P 2 -that play crucial roles. Thus, developing methods for generating asymmetric vesicles containing these specific lipids in a PC background and investigating their impact on pore formation kinetics could reveal mechanistic insights into this unusual process of protein translocation across a membrane.