Figure 1
M6a is present in peripheral fluids. (A) M6a detection in rat cerebrospinal fluid (CSF). Lane 1: Hippocampal lysate, used as a positive control; Lane 2: COS-7 cells lysate (a cell line that do not express M6a endogenously), used as a negative control for M6a expression; Lane 3: Molecular Weight Marker (MW); Lane 4: rat cerebrospinal fluid. Bands representing M6a are indicated by stars. (B) M6a detection in mouse sera depleted from albumin protein. Two (i and ii) independent samples of two mice are shown. Lane 1: Neuron lysate, used as a positive control; Lane 2: Molecular Weight Marker (MW); Lanes 3 and 5: Supernatants (SN) obtained after albumin removal. These lanes show no M6a signal. Lanes 4 and 6: Corresponding pellets (P) obtained after albumin removal. Most minor proteins remain in pellets. Bands representing M6a are indicated by stars. As showed in A and B, M6a migrates as multiple bands due to post-translational modifications of the protein. M6a can be detected as four bands, as a doublet or as one intense single band depending on running conditions, protein load, membrane exposure and sample origin and processing. Full-length blots are shown in Figure S8. (C) EVs isolated from serum samples, observed by transmission electronic microscopy (TEM) negative staining. (D) Immunogold stain (18-nm particles) without (control, 1) or with monoclonal M6a antibody (2 and 3). Note the particles bound to the EV surface. A single EV (2) as well as a cumulus of EVs (3) are shown. (E) Serum-isolated EVs stained for the positive EV marker CD63 and revealed with a secondary antibody conjugated to Alexa-488 (green) or stained for M6a and revealed with a secondary antibody conjugated to rhodamine (red). Magnifications show a CD63-positive EV (green, 1- upper), a double labelled EV (orange, 2-middle) and an M6a-positive EV (red, 3- lower).
