Nbp1 81586

Mouse brains were dissected, fixed in 4% paraformaldehyde, cryoprotected, embedded in OCT, and frozen. Cryostat brain sections (8 μm) were mounted on positively charged slides. Tissue-containing slides were incubated with phospho-cPLA2 -S505- (2831, CST), iPLA2 (NBP1–81586, Novus), and NeuN (NAB377, Millipore) primary antibodies overnight at 4 °C and incubated with secondary antibody (Goat anti-mouse Alexa Fluor® 555, Goat anti-rabbit Alexa Fluor® 647) for 1 h at room temperature, followed by the addition of DAPI-containing mounting media (Vectashield, Vector Laboratories). Images were taken using 20× and 40× objectives on a Nikon A1R VAAS inverted confocal microscope and analyzed using NIS-Elements imaging software (Nikon).

Pulverized cerebrum tissues were homogenized in 1X NP40 on a cooling tissue homogenizer (Cryolys Precellys Evolution Homogenizer). NP40 homogenates were centrifuged at 12,300 rpm for 20 min at 4 °C and supernatants were run into NuPage 4–12% Bis-Tris (Life Technologies, NY) under reducing conditions. Samples were normalized based on total protein content, which was estimated by the BCA protein assay. Western blot analyses were performed using antibodies against cPLA₂ (sc-454 and sc-376,636, Santa Cruz Biotechnology -SCB-), phospho-cPLA₂ -S505- (2831, Cell Signaling Technology -CST-), iPLA₂ (sc-376,563, SCB; and NBP1–81586, Novus), MAPK p42/p44 (4695, CST), phospho-MAPK p42/p44 -T202/Y204- (4370, CST), MAPK p38 (8690, CST), phospho-MAPK p38 (rabbit polyclonal, CST), SAPK/JNK (9252, CST), active JNK (V7931, Promega), PKCα (2056, CST), PKCδ (9616, CST), PKCλ (610,207, BD Biosciences), phospho-PKC pan -βII Ser660- (9371, CST), phospho-PKCα/βII -T638/641- (9375, CST), phosphor-PKCδ -T505- (9374, CST), phospho-PKCζ/λ -T410/403- (9378, CST), CaMK2 (sc9035, SCB), pCaMK2 (sc-12,886-R, SCB), β-Tubulin (2146, CST), GAPDH (MAB374, Millipore), VDAC (4866, CST). Relative intensities were quantified using ImageJ software.