Fig. 1

Biochemical characterization of M18 splice variants. a Alignment of M18L/S C-terminal amino acid (aa) sequences. Mismatching residues are in red. Amino acids highlighted (in yellow) represent the immunogenic sequences used for production of variant-specific antibodies. Omitted M18L/S N-terminal sequences are 100 % identical. b–c Immunoprecipitation (IP) of M18L/S with variant specific antibodies (and anti-mouse IgG as a negative control) using human brain homogenates. NCI control subjects (n = 3) were tested with similar results. IP products, along with input samples and negative controls (IgG), were resolved by (b) standard SDS- or (c) BN-PAGE, followed by either silver staining (Silver) or immunoblotting (IB) with specific antibodies against M18 variants, syntaxin-1 (STX1), SNAP-25 (S25), or VAMP. c M18S antibody recognized two bands at ~150 and ~70 kDa, putatively corresponding to a SNARE-M18 heterotetramer (pointed with a red arrowhead) and the monomeric form, respectively. d, f Schematic illustration depicting the sequential extraction of (d) synaptosomes and (f) lipid rafts from human cortical homogenates. g, relative centrifugal forces; IF, interface; P, pellet; S, supernatant. e Equivolumetric amounts of fractions obtained in (d) were resolved by SDS-PAGE and immunoblotted using antibodies targeting M18L/S, STX1, S25, VAMP, and markers for synaptic vesicles (synaptophysin [SYP]), nuclei (FosB), nuclei + cytosol (NeuN), and myelin fragments (myelin basic protein [MBP]). Note that MBP strongly labels P1, as heavy myelin fragments precipitate along with cell debris. g Equivolumetric amounts of fractions obtained in (f) were resolved by SDS-PAGE and immunoblotted using the antibodies above. α-Synuclein (α-syn) antibody was additionally used, showing similar distribution across fractions as previously reported [66]. e, g IF1 and S4 fractions were framed in a red, dashed box to highlight synaptosome and lipid raft-enriched proteins, respectively. b–g Masses (in kDa) of prestained markers are indicated on the left side of immunoblots