Fig. 9

rE4 and Aβ42 induce greater cPLA2 translocation to synaptic vesicles (cytosolic fraction) in human synaptosomes than rE3. A Frozen postmortem human cortex samples were prepared to collect the supernatant (P1 fraction) and pellets (P2 fraction, synaptosomes) and the different cell type markers were measured by WB. B cPLA2 expression was detected in different fractions. C The LDH assay was performed to test the membrane integrity of the isolated synaptosomes after incubation for 1 hour at different temperatures. D One group of synaptosomes was treated with KR alone (control), and the remaining groups were treated with rE3, rE4, Aβ42 and C-1-P for 30 minutes, followed with C-1-P treatment for 15 minutes. After treatment, the cytosolic and membrane fractions of synaptosomes were isolated by centrifugation. cPLA2 was enriched by immunoprecipitation with anti-cPLA2 antibody and then phosphorylated and total cPLA2 were measured by WB. A representative blot for total cPLA2 is shown. Phosphorylated cPLA2 levels were low and not shown. E Quantification of cPLA2 protein expression in the cytosol fraction. cPLA2 was normalized to β-actin for each group and then adjusted to KR alone for all groups. F Quantification of cPLA2 protein expression in membrane fraction. cPLA2 protein expression was normalized to Na,K ATPase for each group and then adjusted to KR alone for all groups. For E and F, data was obtained from 7 independent repeats. WB. Western blot; C, cytosol; M, Membrane; rE3, recombinant ApoE3; rE4, recombinant ApoE4; C-1-P, Ceramide-1-phosphate; KR, Krebs Ringer buffer. Data presented as mean ± SEM and analyzed by Student’s t-test (two-tailed). *p < 0.05