Figure 1.

Endophilin B1 was localized to EEA1-positive early endosomes. A, Expression of endophilin B1 was developmentally regulated in the brain (top) and SCGs (bottom). Expression of endophilin B1 increased throughout development, with maximal expression detected in the adult stage (Ad). B, Endophilin B1 was localized to light membrane and synaptic vesicle-enriched fractions. Adult brains were subjected to differential centrifugations to obtain various fractions. Immunoblotting against endophilin B1 revealed that endophilin B1 was localized to a light membrane fraction (P3), which includes plasma membrane and intracellular organelles such as endosomes and lysosomes. In addition, endophilin B1 was enriched in a synaptic vesicle-enriched fraction (LP2), indicating that endophilin B1 may also be present in synaptic vesicles. Endophilin A (EndoA) was included as controls. C, Endogenous endophilin B1 colocalized partially with EEA1 and LAMP2 in the cell body of hippocampal neurons, but punctate colocalization was only observed between endophilin B1 and EEA1 in the neurites. Quantitation of the colocalization between endophilin B1 staining and EEA1 or LAMP2 immunoreactivities is depicted in the histogram. Error bars indicate SD. Scale bar, 20 μm. D, Endophilin B1 interacted with EEA1, but not Rab4, Rab7, and LAMP2 in PC12 cells. Association between EEA1 and endophilin B1 was enhanced after 30 min of NGF treatment. Endophilin B1 also interacted with TrkA both before and after NGF treatment, although NGF treatment further enhanced the association.