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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 3. — Gab2 (Grb2-associated binder2) silencing blocks TNFα (tumor necrosis factor alpha)-induced, IL (interleukin)-1β–induced, and lipopolysaccharide (LPS)-induced signaling in the endothelial cells. A and B, Human umbilical vein endothelial cells (HUVECs) transfected with Gab1 (Grb2-associated binder 1) or Gab2 siRNA or scrambled siRNA (scRNA) were serum starved overnight and treated with TNFα (10 ng/mL) for the indicated times. The cell lysates were subjected to immunoblot analysis to probe for the phosphorylated (p) ERK (extracellular signal-regulated kinase) 1/2, JNK (c-Jun N-terminal kinase) 1/2, p38, Akt (protein kinase B), c-Jun, and NF-κB (nuclear factor kappa B; p65) using phospho-specific antibodies. Immunoblots were also probed for total (t) ERK1/2, p38, JNK1/2, or GAPDH for loading controls. Band intensities at the 20 min time point were quantified by densitometry, and these data are shown in the right. C, HUVECs transfected with Gab1 or Gab2 siRNA or scrambled siRNA were treated with TNFα (10 ng/mL), IL-1β (10 ng/mL), or LPS (500 ng/mL) for 30 min. The cell lysates were analyzed for the activation of p38 and NF-κB (p65) by immunoblot analysis. Band intensities were quantified by densitometry, and these data are shown in the right. D, Mouse brain endothelial cells isolated from Gab2^−/− or WT (wild type) littermate control mice (Gab2^+/+) were serum starved overnight and treated with TNFα (20 ng/mL), IL-1β (20 ng/mL), or LPS (1 µg/mL) for 30 min. The cell lysates were evaluated for the phosphorylation of ERK1/2 by immunoblot analysis. Band intensities were quantified by densitometry, and the quantified data are shown in the right. Student t test was used to calculate statistically significant differences for data shown in A and B. For others, 1-way ANOVA was used to compare the data of experimental groups, and Tukey post hoc multiple comparison test was used to obtain statistical significance between the two groups. ns indicates no statistically significant difference. ***P<0.001. Con indicates control vehicle.