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. 2020 Jul 28;142(12):1190–1204. doi: 10.1161/CIRCULATIONAHA.120.048191

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© 2020 American Heart Association, Inc.

This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

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Figure 2. — MDM2 decreases ACE2 stability through ubiquitination. A through C, Western blot analysis of ACE2 and MDM2 levels in PAECs transfected with MDM2 or control siRNA (20 nmol/L) for 48 hours (A); JNJ-165 (10 μmol/L) or vehicle for 16 hours (B); and MDM2 overexpression or control plasmid (1 μg/mL) in BAECs for 48 hours (C). D and E, Western blot analysis of ACE2 in HEK 293 cells transfected with MDM2 siRNA (20 nmol/L), MDM2 overexpression plasmids (MDM2-WT), or MDM2 RING domain mutation plasmids (MDM2-C464A) together with ACE2 overexpression plasmids (1 μg/mL), then treated with CHX (100 μg/mL) for up to 8 hours. F, HEK 293 cells were cotransfected with ACE2 and ubquitin (Ub) overexpression plasmids (1 μg/mL) and were treated with MG132 (20 μmol/L) 10 hours before harvesting the cells. Separately, HEK 293 cells were treated with JNJ-165 (10 μmol/L) or vehicle for 16 hours before harvesting (a). HEK 293 cells were treated with MDM2 or control siRNA (20 nmol/L) and cultured under a normal or hypoxia condition (b and c) for 24 hours. Cell extracts were immunoprecipitated with anti-ACE2 or IgG and immunoblotted with anti-Ub. The input proteins were immunoblotted with antibodies against ACE2, MDM2, or α-tubulin. G, HEK 293 cells were transfected with plasmids overexpressing ACE2 or MDM2 (1 μg/mL). H, HUVECs were treated with MG132 (20 μmol/L) for 10 hours. ACE2 or MDM2 was immunoprecipitated. The input protein was immunoblotted with antibodies against ACE2, MDM2, α-tubulin, or GAPDH. Data are mean±SEM from 3 to 5 independent experiments. Normally distributed data (B) were analyzed by 2-tailed Student t test with Welch correction between 2 indicated groups and normally distributed data (D and E) were analyzed by 2-way ANOVA test between multiple groups. Nonnormally distributed data (A and C) were analyzed using the Mann-Whitney U test between 2 indicated groups. *P<0.05 versus controls (Ctrl) or control-siRNA (Ctrl-siR). ACE2 indicates angiotensin-converting enzyme 2; BAEC, bovine aortic endothelial cell; CHX, cycloheximide; HEK 293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cell; IB, immunoblotted; IgG, immunoglobulin G; IP, immunoprecipitation; MDM2, murine double minute 2; PAEC, pulmonary artery endothelial cells; siR, siRNA, small interfering RNA; and WT, wild type.

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