Anti phospho marcks

HEK293 cells were lysed in mammalian protein extraction reagent (MPER, Thermo Fisher Scientific, Waltham, MA, United States) containing Halt protease and phosphatase single use inhibitor (Thermo Fisher Scientific, Waltham, MA, United States). Lysates were separated by SDS-PAGE using Criterion TGX gels (Bio-Rad, Berkeley, CA, United States), and transferred onto 0.2 μM PVDF membranes (Bio-Rad, Berkeley, CA, United States). Membrane were blocked with 5% non-fat milk in TBS for 1 h, incubated with anti-TREM2, anti-DAP12, anti-Phospho-Akt (Ser473), anti-AKT, anti-IκBα, anti- Phospho-NF-κB p65 (Ser536), anti-NF-κB p65, anti-Phospho-MARCKS (Ser167/170), anti-MARCKS, anti-Actin (1:1000, Cell Signaling, Danvers, MA, United States), anti-Phospho-Syk (Tyr525/526) (1:1000, Abgent, United States), or anti-SYK (4D10, 1:1000, Santa Cruz, CA, United States) primary antibody overnight at 4°C, and incubated in HRP-conjugated anti-Rabbit or anti-Mouse secondary antibody (1:3000, Cell Signaling, Danvers, MA, United States) at room teMPERature for 1 h. Western blots were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, United States). Signals were quantified using ChemiDoc XRS (Bio-Rad, Berkeley, CA, United States) and densitometric analysis were performed using Image Lab (Bio-Rad, Berkeley, CA, United States).

Synaptosomes were lysed in an ice-cold Tris-HCl buffer solution and then centrifuged for 10 min at 13,000× g at 4 °C. Protein content was determined by using the Bradford assay. Equal amounts (30 µg) of samples were loaded per lane onto 10% polyacrylamide gel, and then transferred to a polyvinylidenedifluoride (PVDF) membrane in a semi-dry system (Bio-Rad, Hercules, CA, USA) for 120 min at a constant current of 0.15 mA. Membranes were blocked with Tris-buffered solution that contained 4% bovine serum albumin for 1 h at room temperature under agitation. After blocking, membranes were incubated overnight at 4 °C with the primary antibodies (anti-protein kinase C (PKC), 1:700; anti-phospho-PKC, 1:2000; anti-protein kinase C alpha (PKCα), 1:600; anti-phospho-PKCα, 1:2000; anti-phospho-MARCKS, 1:250; anti-β-actin, 1:1000; Cell Signaling Technology, Beverly, MA, USA). The immunoreactive bands were visualized by using peroxidase-conjugated donkey anti-rabbit IgG secondary antibodies (1:1000; Cell Signaling Technology, Beverly, MA, USA) and enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK). After protein detection, densitometric analyses were performed using ImageJ software.