Electrotransfer system

The BV-2 cells were homogenized with RIPA buffer and protease inhibitors (Thermo Scientific). Equal amounts of protein (40 μg/lane) were loaded to gradient (4 to 15%) concentrations of polyacrylamide SDS-PAGE (Smobio, Hsinchu City, Taiwan) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) using an electro-transfer system (Bio-Rad, Hercules, CA). The membranes were blocked with 5% nonfat milk and probed with the following primary antibodies for 16 h at 4 °C: rabbit polyclonal anti-CD206 (1:1,000; Abbkine, Wuhan, China), goat polyclonal TREM2 (1:1,000, ThermoFisher Scientific), and mouse monoclonal β-actin (1:5,000; Abbkine). The membranes were incubated with the appropriate secondary antibodies (Bethyl, Inc., Montgomery, TX, USA) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Bound antibodies were visualized with D-Plus ECL Pico System (Dongin LS, Republic of Korea) and a G: Box Chemiluminescence & Fluorescence system (Syngene, Frederick, MD, USA).

The total protein content from the cells was extracted in cell RIPA buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate, supplemented with a mixture of complete protease inhibitors and phosphatase inhibitors. After centrifugation, the protein in the supernatant was collected and stored at −20 °C. The protein samples were determined using the Lowry protein assay. Briefly, equal amounts (20–30 μg) of protein were heated after adding the appropriate amount of 5× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (40% glycerol, 10% SDS, 5% 2-mercaptoethanol, 0.02% bromophenol blue, 250 mM Tris–HCl, pH 8.8). The samples were separated on 6%–15% SDS-PAGE and subsequently transferred onto nitrocellulose filters using the Bio-Rad electrotransfer system (Bio-Rad Laboratories, Munich, Germany). The blots were then incubated with specific primary antibodies (1:1000) overnight at 4 °C, washed three times with TBST, and incubated with the appropriate secondary antibody for 1 h at room temperature. β-Actin, LATS2, Ki-67, α-SMA (Abcam, Cambridge, United Kingdom), ERK1 (BD Biosciences), p-Akt, p-ERK1/2, Akt, p21, p27, α-tubulin, HOAC1, and FOXC1 (Santa Cruz Biotechnology) primary antibodies were used. Finally, the blots were developed using a custom-made ECL detection system.

40 μg proteins per sample were separated using 4–20% SDS-PAGE and transferred to 0.2 µm nitrocellulose Trans-blot turbo^TM membranes (Bio-Rad Laboratories Inc.) using the Bio-Rad electrotransfer system (Bio-Rad Laboratories Inc.). The membranes were blocked with blocking buffer (mixed 5% non-fat dry milk, 150 mM NaCl, 0.1% Tween-20 and 20 mM Tris-HCl and adjusted to a pH of 7.6) for 1 h at room temperature and probed with specific primary antibodies at 4 °C overnight. The protein bands were detected with HRP-conjugated secondary antibodies for 1 h at room temperature using custom-made ECL^TM Prime Western Blotting Detection Reagents (Amersham GE, Little Chalfont, UK). The Image Lab^TM software digital imaging system ChemiDoc^TM XRS+ (Bio-Rad Laboratories Inc.) was used to detect the target protein on immunoblot nitrocellulose membranes.