cPLA2 activity measurement

cPLA2 activity in brain tissue was determined using the cytosolic phospholipase A2 assay kit from Abcam (ab133090) according to manufacturer’s instructions. Briefly, approximately 5 mg of mouse brain tissue were homogenized in 200 μL HEPES/EDTA cold buffer (50 mM HEPES, pH 7.4, 1 mM EDTA) with a probe sonicator in an ice water bath. After centrifugation at 10,000 × g for 15 min at 4 °C, 200 μl of each supernatant was concentrated using 10 K molecular weight cut-off concentrators (Thermo Fisher Scientific, San Jose, CA). sPLA2 and iPLA2 have been removed by membrane filter and inhibited by bromoenol lactone respectively according to manufacturer’s instructions. Each sample (10 μl) was used to determine cPLA2 activity after 60 min of reaction. The final absorbance at 414 nm was read using a plate reader. The cPLA2 activity was normalized by protein concentration. Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific, San Jose, CA).