Pierce direct magnetic ip co ip kit

Cell surface proteins of hCMEC/D3 were biotinylated according to the manufacturer's instructions (Pierce Cell Surface Protein Isolation Kit; Pierce Biotechnology, Rockford, IL, USA). Briefly, hCMEC/D3 were washed twice with ice-cold PBS and labeled with sulfo-NHS-SS-biotin at 4°C for 30 min. Cells were collected, washed with TBS and lysed in IP Lysis Buffer (Pierce™ Direct Magnetic IP/Co-IP Kit; Pierce Biotechnology, Rockford, IL, USA) with protease inhibitors (Halt™ Protease Inhibitor Single-Use Cocktail; Pierce Biotechnology, Rockford, IL, USA) by incubating on ice for 30 min with gentle vortexing every 10 min. The cell lysate was centrifuged at 13,000 rpm for 20 min. The supernatant was collected and protein concentration was measured by bicinchoninic acid (BCA) protein assay (Pierce BCA Protein Assay Kit; Pierce Biotechnology, Rockford, IL, USA). The biotinylated cell surface proteins were isolated via magnetic beads (Pierce™ Direct Magnetic IP/Co-IP Kit; Pierce Biotechnology, Rockford, IL, USA) coupled to anti-biotin antibody (Rabbit Anti-Biotin Antibody ab53494; Abcam Inc., Cambridge, MA, USA). The proteins eluted from the magnetic beads were quantified with Bradford protein assay (Quick Star™ Bradford Protein Assay; Bio-Rad Laboratories Inc., Hercules, CA, USA).

Briefly, cells were lysed with an IP lysis buffer (Beyotime, Shanghai, China), and the protein concentration was quantified by a BCA Protein Assay Kit (Beyotime, Shanghai, China). Co-IP assays were performed using a Pierce Direct Magnetic IP/Co-IP kit (Thermo Fisher Scientific, Rockford, United States) in accordance with the manual. The proteins were eluted in a 2 × loading sample buffer and then boiled for 10 min at 100°C. The subsequent detection steps were as described for immunoblotting analysis. The antibodies used are presented in Supplementary Table 2.

Co-IP assays were performed using a Pierce Direct Magnetic IP/Co-IP kit (Thermo Scientific, Rockford, IL) according to the manual. Briefly, cells were incubated with lysis buffer on ice for 5 min with periodic mixing. The lysis solution was quantified and diluted. Five hundred microliters of lysate was then incubated with 25 µl of beads coupled with 5 µg of antibody at 4 °C overnight. After extensive washing, the beads were spun down, resuspended and boiled. The pull-down samples were detected using western blotting according to standard protocols.