Wnt3a-induced exosome secretion occurs independently of GSK3 activity in microglia. Microglia were pre-treated with DKK1 (30 nM) or lithium (5 μM) for 1 hour before the addition of Wnt3a for 8 hours. Exosomes were subsequently prepared by centrifugation of the cell culture medium at 10000xg for 10 minutes, followed by concentration of the medium using 3kDa centrifugal devices before exosomes were isolated at 100000xg. The exosomal fractions were subjected to western blotting for the detection of β-actin as a marker of exosomal abundance (A). Lanes 3 and 4 depict: Wnt3a + inhibitor and the effect of the inhibitor alone respectively. The TOPflash assay in HEK293a cells illustrates the effects of Wnt3a, Wnt3a + DKK1, DKK1, and lithium on β-catenin dependent signaling (B). ** p < 0.01.
Hooper et al. BMC Neuroscience 2012 13:144 doi:10.1186/1471-2202-13-144