Fig 1: RegDCs EXO protect encapsulated immunoregulatory cargo (TGFB1 and IL10) from proteolytic degradation. (A) Nano-tracking analysis to determine EXO number and size distribution in nm. (B) Transmission electron microscopy (TEM) to visualize EXO shape and immuno-gold TEM to detect EXO marker tetraspanin CD63, showing unstained (left) and positive staining (right)(arrows). (C) Western blotting to detect other EXO-related markers including CD81, TSG101, GRP94 and B-actin in donor DCs and EXO (left) and anti/pro-inflammatory cytokines including TGFB1, IL10, IL6, IL1B and TNF and the costimulatory molecule CD86 as well as EXO associated proteins ALIX and TSG101 in DCs EXO subsets (right). (D) TGFB1 and IL10 content of lysed EXO and in regDCs EXO supernatant by ELISA. (E) TGFB1 and IL10 content of non-lysed EXO (transmembrane domain) by ELISA. (F) Immunogold TEM to detect luminal and transmembrane TGFB1 in regDCs EXO. (G) regDCs EXO (left) or equivalent concentration of free TGFB1 and IL10 (right) were treated with trypsin (upper panel) or proteinase-K (lower panel) and incubated in control buffer (1hour at 37°C) and analysed by western blotting to detect the levels of TGFB1, IL10 and exosomal markers TSG101 (upper panel) and CD81 (lower panel).