FIGURE 6:

Acute perturbation of clathrin function does not prohibit synaptic-like microvesicle formation after release of a brefeldin A block. PC12 cells stably expressing mCh-FKBP-CLC were treated for 2 h with brefeldin A (BFA) or vehicle control (EtOH) at 37°C. After this 2-h treatment, BFA-treated cells were either transferred to 4°C (BFA) or extensively washed at 4°C and then incubated with AP21087 (BFA-AP20187) or vehicle control (BFA-EtOH) for additional 2 h at 37°C. SLMVs sediment to the middle of 5–20% glycerol velocity gradients. Fractions from this gradient were loaded onto SDS–polyacrylamide gels for Western blot and densitometry quantifications. Immunoblot (IB) for SV2 and VAMP7 was used to track SLMVs. (A, A′) Densitometry quantification of fraction signal per total signal in BFA-treated cells (white circles) as compared with EtOH-only treated cells (black circles). (B, B′) Densitometry quantification of fraction signal per total signal in BFA-EtOH–treated cells (black circles). (C, C′) Densitometry quantification of fraction signal per total signal in BFA-treated cells (white circles) as compared with BFA-AP20187–treated cells (black circles). (D, E) Western blot of VAMP7 and SV2, respectively, from EtOH-, BFA-, BFA-EtOH–, and BFA-AP20187–treated cells.