Proteoextract native membrane protein extraction kit

Membrane fractionation was performed using the Proteoextract Native Membrane Protein Extraction Kit (Millipore, 444810) according to the manufacturer's instructions.

For the cell membrane protein extraction, all cell types were seeded in 15 cm plates. When they reached confluence, cells were harvested using a scraper in cold PBS and isolated by centrifugation at 700 g for 5 min. The supernatant was aspirated, and the cell pellets were frozen at −80 °C. The ProteoExtract^® Native Membrane Protein Extraction Kit (Millipore, USA) was employed following the manufacturer’s instructions to obtain nondenatured functional membrane proteins. In brief, the cell pellet was washed twice with the washing buffer, and then incubated with ice-cold Extract Buffer I at 4 °C for 10 min under gentle agitation. The pellet was centrifuged at 16,000 g for 15 min (4 °C). The supernatant was discarded, and 1 mL ice-cold Extract Buffer II was added to the pellet. This membrane protein extraction step was allowed for 30 min at 4 °C under gentle agitation. Then the supernatant was collected after centrifugation at 16,000 g for 15 min at 4 °C. The membrane extracts were characterized with a BCA Protein Assay kit (Takara, Dalian, China) and then stored at −80 °C for SiNR coating.

HBMEC cells were lysed in lysis buffer in the presence of Protease Inhibitor Cocktails (Thermo). Total proteins were harvested after insoluble materials were removed by centrifugation at 14,000 rpm for 20 min at 4°C. Membrane proteins of HBMECs were extracted using ProteoExtract Native membrane Protein Extraction Kit (Millipore). Equal amounts of protein (20 μg/lane) were separated in 4–12% NuPAGE Bis-Tris gels (Invitrogen), and then transferred onto nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% non-fat dry milk and then probed with primary antibodies against VE-cadherin (Abcam), claudin-5 (Abcam), occludin (Abcam), ZO-1 (Abcam), and Na-K-ATPase (Abcam) at 4°C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature, followed by an enhanced chemiluminescent substrate for detection of HRP (Pierce). The optical density of protein bands was quantified using NIH ImageJ.

HEK293T and HEK293T ZAP KO cells (0.6–0.8 mln) were seeded in 6-well plates. The following day, ZAP KO cells were co-transfected using PEI MAX with 60 ng pcDNA HA-ZAP constructs and 940 ng pcDNA3.1 empty vector. Cells were harvested two days later, washed in PBS and processed using ProteoExtract Native Membrane Protein Extraction Kit (Sigma). Soluble cytoplasmic, membrane protein and insoluble fractions were isolated according to the manufacturer’s instructions, with the addition of three 1 ml PBS or high salt washes between extraction buffer I and II. The insoluble debris fraction was resuspended in RIPA buffer, sonicated and reduced in Laemmli buffer by boiling at 95°C for 10min.