Am926

Tissue or cell lysates containing 20 μg of protein were prepared, and electrophoresed on a 10% SDS-PAGE gel under denaturing conditions, subsequently transferred to a PVDF membrane by electroblotting. The membrane was blocked in 5% nonfat milk for 60 min at room temperature, then incubated with the primary antibodies overnight at 4 °C. After washing with TBST three times, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (BOSTER,CA,USA) for 120 min at room temperature. The antibodies used included anti-PLOD2 mouse monoclonal antibody (1:3000 dilution; mab4445, R&D); anti-CD44, anti-CD133, anti-β-catenin rabbit polyclonal antibody (1:3000 dilution; 9582, CST), anti-myc mouse monoclonal antibody (1:3000; AM926, Beyotime), anti-MDR1 rabbit polyclonal antibody (1:3000; BA1351, BOSTER), anti-MRP antibody, anti-caspase-3 mouse monoclonal antibody (1:3000; SC-7272, SANTA), α-tubulin monoclonal mouse monoclonal antibody (1:3000 dilution; T9026, Sigma-Aldrich), and anti-GAPDH antibody (1:3000 dilution; BM1623, BOSTER).

For the Co-IP analysis, HEK-293 T cells were cotransfected with 1 µg/ml pCMV-ACE2-MYC and pCMVPuro05-Control, pCMVPuro05-ApoE2-3 × Flag, pCMVPuro05-ApoE3-3 × Flag or pCMVPuro05-ApoE4-3 × Flag. After transfection for 48 h, the cells were washed with PBS and lysed with IP lysis buffer for 30 min at 4 °C. The supernatants of the cell lysates were incubated with the corresponding antibody overnight at 4 °C, incubated with protein A/G agarose beads (Santa Cruz, sc-2003) for 2 h at 4 °C, and washed three times with lysis buffer. The proteins were analysed by western blotting using anti-Flag (1:1,000, Abbkine, A02011) and anti-MYC (1:1,000, Beyotime, AM926) antibodies.