FIGURE 1.
EV‐associated MICA*008 and not its soluble form induces NKG2D downmodulation. (a) Conditioned supernatants derived from ARK MICA*008 transfectants or ARK transfected with an empty vector were depleted of EVs and used to culture the NKL cell line for 24 h. NKG2D and DNAM‐1 expression was evaluated by immunofluorescence and FACS analysis. NKG2D relative expression was calculated respect to the untreated group (= 100%) represented by dashed line. Values reported represent the mean of independent experiments. (b‐f) ARK cell transfectants were cultured for 48 h in EV‐free medium and then EVs were isolated from the conditioned media as described in materials and methods. (b) Size distribution of sEVs and mEVs was analysed through dynamic light scattering (DLS). A representative experiment is shown. (c) Transmission electron microscopy (TEM) of sEVs and mEVs preparations. Bar corresponds to 200 nm. (d) The amount of total proteins recovered from EV preparations derived from 106 cells was calculated by BPA and corresponded to 1.127 μg ± 0.58 for sEVs and 3.04 μg ± 1.79 for mEVs. (e) The number of EVs/μg of protein was calculated through DLS. (f) Western blot analysis was performed on lysates (40 μg) derived from sEVs and mEVs fractions or from cell pellet, using anti‐Hsp70, anti‐calreticulin, anti‐CD63, anti‐MHC I and anti‐CD81 antibodies