Anti hemagglutinin ha

Immunoblot analysis was performed as previously described (44 (link)). Antibodies were used as follows: anti-USP25 (Abcam, Cambridge, MA, USA; ab187156, 1:1000), anti-APP (Millipore, Billerica, MA, USA; MAB348, 1:1000), anti-Iba1 (Wako Pure Chemical, Osaka, Japan; 016-20001, 1:500), anti-GFAP (glial fibrillary acidic protein) (Cell Signaling Technology, Danvers, MA, USA; 3670, 1:1000), anti–β-III-tubulin (Abcam, ab18207, 1:1000), anti-HA (hemagglutinin) (Sigma-Aldrich, St. Louis, MO, USA; H6908, 1:1000), anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-8017, 1:500), anti–β-actin (Xmbcss, Xiamen, China; bc001, 1:2000), and horseradish peroxidase (HRP)–conjugated secondary antibodies (Thermo Fisher Scientific, 31430 or 31460; 1:3000).

Tissue culture medium was from Gibco. [γ-³²P]ATP was purchased from PerkinElmer. Palmitate, fatty-acid-free BSA, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], ethylene-diamine-tetra-acetic acid (EDTA), disodium salt, perchloric acid, hydrogen chloride, Anti-HA (hemagglutinin), SPT2 antibodies, and insulin human solution were from Sigma–Aldrich. Myriocin, l-cycloserine, D609, C₂-ceramide, C₂-dihydroceramides, and N-butyldeoxynojirimycin were from Enzo Life Sciences. Ro31-8220 and DAG kinase were from Calbiochem. All solvents were from Merck Eurolab or Fisher Scientific. phospho-Akt (Thr 308), phospho-Akt (Ser 473), Akt, p-PKCζ (Thr 410-403), PKCζ, and β-actin antibodies were from Cell Signaling. Ultrapure water was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). Adenoviruses containing the cDNA of GFP, wild-type PKCζ (WT- PKC), constitutive active PKCζ (CA-PKCζ), or kinase-dead PKCζ (KD-PKCζ) were prepared as previously described [14] (link). All PKCζ constructs contained an HA tag for monitoring their expression.

Protein extracts were prepared using a NaOH cell lysis method (Dilova et al., 2002 (link)), loaded onto SDS–PAGE gels, and transferred to nitrocellulose membrane. Membranes were probed with anti-hemagglutinin (HA; 1:5000; Sigma-Aldrich, St. Louis, MO), anti–glucose-6-phosphate dehydrogenase (G6PDH; Zwf1; 1:100,000; Sigma-Aldrich), or anti-GFP (1 μg/ml; N86/8; Neuromab, Davis, CA) primary antibodies and visualized using the appropriate secondary antibodies conjugated to IR Dye (1:5000; Li-COR Biosciences, Lincoln, NE). Quantifications were performed using ImageQuant software (GE Healthcare, Little Chalfont, UK). The relative distribution of the signal in each lane was figured by measuring the level of signal in three distinct portions of each lane—upper (hyperphosphorylated), center (phosphorylated), and lower (dephosphorylated)—and then dividing each portion by the total amount of signal within each lane.