Fig. 6

NEDD4L may inhibit ACE2 expression through ubiquitination. Before Co-IP, HEK293 cells were transfected with ACE2 and ubiquitin-overexpressing plasmids (1 µg/mL) for 48 h, and then, control-siRNA and NEDD4L-siRNA (20 nM) were added for 24 h. After that, HEK293 cells were treated with MG132 for 10 h (A). Before Co-IP, PASMCs were treated with MG132 for 10 h (B). A correlation between NEDD4L and ACE2 was detected in HEK293 cells (A) and PASMCs (B) by co-IP. The mechanism by which NEDD4L-mediated ubiquitination of ACE2 affects PAH (C). Inhibiting the expression of NEDD4L can reduce its ability to bind to ACE2 via ubiquitination, thereby increasing the expression of ACE2, promoting the apoptosis of PASMCs, and alleviating pulmonary vascular remodeling. IgG, immunoglobulin G; IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitin; NEDD4L, neural precursor cell–expressed developmentally downregulated gene 4-like; ACE2, angiotensin-converting enzyme 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PASMCs, pulmonary arterial smooth muscle cells; HEK293, human embryonic kidney 293