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. 2021 Apr 8;41(6):1987–2005. doi: 10.1161/ATVBAHA.121.316153

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© 2021 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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 6. — Gab2 (Grb2-associated binder2) silencing suppresses thrombin-induced signaling and inflammation in endothelial cells. A, Human umbilical vein endothelial cells (HUVECs) were transfected with 200 nmol/L scrambled siRNA (scRNA), Gab1, or Gab2 siRNA. After 48 h, the cells were treated with thrombin (10 nmol/L) for 6 h. The cell lysates were analyzed for VCAM1 (vascular cell adhesion molecule 1) and ICAM1 (intercellular adhesion molecule 1) protein levels by immunoblot analysis. Band intensities were quantified by densitometry, and these data are shown in the right. B, HUVECs transfected with scrambled, Gab1, or Gab2 siRNA were treated with thrombin for 6 h, and the MCP-1 (macrophage chemoattractant protein 1) levels were analyzed in the cell supernatants by ELISA. C, HUVECs transfected with Gab1 or Gab2 siRNA were treated with thrombin for indicated time points, and the activation of ERK (extracellular signal-regulated kinase), p38 MAPK (mitogen-activated protein kinase), and NF-κB (nuclear factor kappa B) was analyzed by immunoblot analysis using antibodies that recognize phosphorylated (p) ERK, p38, or p65. Band intensities were quantified by densitometry, and these data are shown in the right. D, HUVECs were transfected with a scrambled siRNA or siRNA specific for c-Src (cellular Src), Fyn, or Yes. After 48 h, the cells were treated with thrombin for 5 min. Gab2 was immunoprecipitated and probed for tyrosine phosphorylation using the pY monoclonal antibody in immunoblot analysis. Band intensities were quantified by densitometry, and these data are shown in the right. Data were representative of 3 independent experiments with similar results. One-way ANOVA was used to compare the data of experimental groups, Tukey post hoc multiple comparison test was used to obtain statistical significance. ns indicates no statistically significant difference. ***P<0.001, **P<0.01, *P<0.05. Con indicates control; and Unt, untreated.