293T-ACE2 cells were incubated with Spike-Fc (SinoBiological, Cat#40591-V02H), Spike-Fc/APOE3, and Spike-Fc/APOE4 in culture medium for 1 h at 4 °C, respectively. Cells were washed three times in PBS, and then fixed in 4% PFA in PBS for 10 min at RT. Fixed samples were rinsed in PBS,blocked in PBS containing 10% donkey serum for 1 h at RT, and incubated with antibodies against ACE2 or SARS-CoV-2 spike glycoprotein RBD for 12 h at 4 °C. After washing with PBS, cells were incubated with secondary antibodies: star red-conjugated goat anti-mouse (Abberior Instruments, Cat#NC1933868) or star orange-conjugated goat anti-rabbit (Abberior Instruments, Cat#NC1933866) for 1 h at RT. Cell nuclei were labeled with DAPI for 20 min at RT. After PBS washing, samples were mounted in Vectashield mounting medium and imaged with a STEDYCON (Abberior Instruments) on an inverted Nikon microscope.
Zhang H., Shao L., Lin Z., Long Q.X., Yuan H., Cai L., Jiang G., Guo X., Yang R., Zhang Z., Zhang B., Liu F., Li Z., Ma Q., Zhang Y.W., Huang A.L., Wang Z., Zhao Y, & Xu H. (2022). APOE interacts with ACE2 inhibiting SARS-CoV-2 cellular entry and inflammation in COVID-19 patients. Signal Transduction and Targeted Therapy, 7, 261.
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