Fig. 6: MAAP interacts with the surface of extracellular vesicles.
From: The membrane associated accessory protein is an adeno-associated viral egress factor
A HEK293 cells were transfected with expression vectors encoding Rab7-GFP, Rab11-GFP, MAAP8-HA. MAAP-HA was detected by immunofluorescence with an AlexaFlour647 secondary antibody (MAAP8-HA-A647). A Z-stack of confocal optical sections at 1-μm steps was acquired. A 3-μm-thick medial stack is shown. Images are representative of three experiments. Scale bars, 10 μm. B Co-localization between MAAP8-HA and Rab7-GFP or Rab11-GFP in the whole cell as assessed by Pearson’s correlation coefficient (R) as described in Materials and Methods. Each dot represents one cell. Horizontal bars represent the mean ± SEM. A two-sided Mann–Whitney rank test was used to determine significance. (Rab7 + vs Rab11 + , ****p < 0.0001). C Chips coated with separate capture spots for anti-CD81, anti-CD63, and anti-CD9 were used to capture EVs from media of cells expressing CMV-HA and CMV-MAAP8-HA. Total captured EVs on each chip were quantified (n = 3). Data are presented as mean values ± SD. D Representative size distribution profile of tetraspanin-positive EVs determined by label-free interferometry (n = 3). E CD81 capture probe images of EVs captured from media of cells expressing CMV-HA and CMV-MAAP8-HA. EVs were probed with anti-CD81-CF488a (blue), anti-HA-Alexa Fluor 555 (green), anti-CD9-CF647 (red), and anti-CD63-CF647 (red) (n = 3). F Quantification of percent HA labeled EVs captured from media of cells expressing CMV-HA and CMV-MAAP8-HA (n = 3). Data are presented as mean values ± SD. G EVs from media of cells expressing CMV-MAAP8-HA were captured with anti-CD81, anti-CD63, and anti-CD9 coated chips. EVs were fixed, permeabilized and probed with anti-HA-Alexa Fluor 555 to determine the EV loading orientation of MAAP8-HA (n = 3). Data are presented as mean values ± SD. H Cartoon schematic depicting EV surface loading of MAAP.
