Fig. 2

C9orf72 is enriched in synaptosomes. a Immunoblot analysis of total protein lysates of different mouse tissues reveals widespread C9orf72 protein expression detected as single band around 50 kDa with highest expression levels in brain followed by spinal cord. GAPDH is shown as loading control. b No obvious changes are observed between different brain regions by immunoblot analysis of protein lysates from cortex, hippocampus, striatum, cerebellum and spinal cord. GAPDH is shown as loading control. c Immunoblot analysis of nuclear and cytoplasmic protein fractionations extracted from adult mouse brain reveals localization of C9orf72 to the cytoplasm. α-tubulin and Histone H3 are shown to demonstrate purity of the cytoplasmic and nuclear fractions, respectively. d Immunoblot analysis and quantification of C9orf72 expression levels over mouse brain development from P1 to P300 showing increase of C9orf72 between P1 and P16. e Schematic of the purification protocols for synaptic vesicles and postsynaptic densities (PSDs). Mouse forebrains were homogenized and centrifuged to generate a crude synaptosomal fraction (P2). P2 fractions were fractionated into synaptosomal heavy membranes (LP1), synaptic vesicles (LP2), and synaptic cytosol (LS2) by hypotonic lysis and differential centrifugation. Alternatively, P2 fractions were centrifuged through sucrose gradient to reveal a pure synaptosomal fraction which was further processed to isolate pure PSDs. f C9orf72 is detectable in the synaptosomal fraction P2, and is released into the LS2 fraction containing the soluble cytoplasmic content of synaptosomes. g C9orf72 is enriched in the pure synaptosomal fraction, but absent from PSD fractions. f and g The purity of fractions was confirmed with specific marker proteins for synaptic vesicles (synaptophysin), postsynaptic densities (PSD-95), mitochondria (Cox-IV), and lysosomes (Lamp1). MW size marker: PageRule Plus Prestained Protein Ladder (a); Precision Plus Protein Dual Color Standards (b-d, f, g)