Dynamin inhibition affects post-Golgi secretion and movement of apoE-containing vesicles.

(A) HMDM were treated with ±30 µM MiTMAB for 2 h and cellular ApoE glycoform distribution was determined by 2D-GE and Western blotting. (B) CHO-GFP-VSVGt cells were incubated overnight at 40°C to accumulate VSVG in the ER. Cells were then exposed to 30 µM MiTMAB, Dyngo-4a and Dynole-34-2 for indicated time points at 32°C. Cell lysates were harvest and cleavage of VSVGt by Endo H assessed as described in Material and Methods. GFP-VSVGt (≈120 kDa) and α-tubulin (52 kDa) as a loading control are shown for each time point. (C) HMDM were transiently transfected with apoE-GFP and cultured for 24 h prior to performing live cell microscopy. Individual cells expressing apoE-GFP were identified using a Zeiss LSM 780 microscope equipped with a heated stage and CO2 chamber, and GFP-positive vesicles were tracked for 3–5 min. HMDM were then treated with 30 µM MiTMAB or Dynole-34-2 for 15–30 min prior to imaging. Representative movies are shown in the Supplement. Average speed was quantified using Imaris software and 20 representative spots were randomly selected from 7 cells from at least 3 individual donors. *Pv Ctrl.