Mem per plus protein extraction kit

Cytoplasmic and plasma membrane fractions were prepared from 6×10⁶ cells using the Mem-PER Plus Protein extraction kit (Thermo Fisher, #P-189842). Whole cell lysates or equivalent amounts of fractionated extracts were resolved on polyacrylamide gel, transferred to nitrocellulose membrane and stained with monoclonal TREM2 Abs that recognize C-terminal epitope (Cell Signaling Technology, #91068) at 1:1,000 dilution. For gel loading and cell fractionation controls, we used $\text{β}$ -Tubulin (Cell Signaling Technology, #86298, 1:1,000), HRP-conjugated GAPDH (BioLegend, clone W17079A, 1:4,000) and CANX Abs (BioLegend, #699401, 1:2,000). Secondary Abs were: HRP donkey anti-rabbit IgG (BioLegend, #406401, 1:1,000), HRP goat anti-rat IgG (sc-2006, Santa-Cruz, 1:2,000) and HRP goat anti-mouse IgG (Abcam, ab97023, 1:10,000).

Confluent ECs were made quiescent in serum-free DMEM for 12 h before specified agents were added. The Mem-Per Plus protein extraction kit (ThermoFisher Scientific, Waltham, MA; Catalog No. 89842) was used to isolate cell membrane proteins, following the manufacturer’s protocol with minor modifications. Briefly, the ECs were scraped from the plates with cell wash solution. The cell suspension was centrifuged at 2000 x g for 5 min. The supernatant was discarded and the pelleted cells were washed twice with Cell Wash Solution. Permeabilization Buffer was added and the cells were permeabilized while mixing continuously at 4°C for 20 min. An aliquot was separated for later use to determine total target protein levels. The permeabilized cells were centrifuged at 16,000 x g for 15 minutes. The supernatant containing the cytosolic proteins was removed without disrupting the pellet containing the membrane fraction. Solubilization Buffer was added to the membrane fraction and the sample was mixed for 45 min at 4°C. After centrifuging the sample at 16,000 x g for 15 min, the supernatant containing those solubilized membrane proteins was collected and stored at −80°C for later use. To determine total target protein level, the aliquot separated after the cells were permeabilized was mixed with lysis buffer. Total target protein level was determined by immunoblot analysis as described above.