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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 4. — TNFα (tumor necrosis factor alpha), IL (interleukin)-1β, and lipopolysaccharide (LPS) induce the phosphorylation of Gab2 (Grb2-associated binder2) and association of Gab2 with its interacting partners, SHP2 (SH2 containing protein tyrosine phosphatase-2) and PLCγ (phospholipiase C gamma). A, Human umbilical vein endothelial cells (HUVECs) were treated with TNFα (10 ng/mL), IL-1β (10 ng/mL), or LPS (500 ng/mL) for the indicated times. The cells were lysed in the radioimmunoprecipitation assay buffer containing protease inhibitors. Gab2 was immunoprecipitated (IP) using the Gab2 polyclonal antibody, followed by protein A/G agarose bead pull-down. The Gab2 immunoprecipitates were subjected to immunoblot analysis (IB) and probed with a phosphotyrosine (pY)-specific monoclonal antibody to evaluate the phosphorylation of Gab2. Band intensities were quantified by densitometry, and these data are shown in the right. B, HUVECs were treated with the TNFα (10 ng/mL), IL-1β (10 ng/mL), LPS (500 ng/mL), or a control vehicle for 5 min. The Gab2 was immunoprecipitated, and the immunoprecipitates were probed for SHP2, PLCγ, or P85α by immunoblot analysis using specific monoclonal antibodies against SHP2, PLCγ, or P85α. C, HUVECs were incubated with Src inhibitor PP2 (10 µmol/L) or its inactive analog PP3 (10 µmol/L) for 1 h. Then, the cells were treated with the agonists for 5 min. The Gab2 was immunoprecipitated and probed with a pY monoclonal antibody to evaluate the tyrosine phosphorylation of Gab2. D, HUVECs were transfected with 200 nmol/L of scrambled siRNA (scRNA) or siRNA specific for c-Src, Fyn, or Yes. After 48 h, the cell lysates were harvested and probed for expression of c-Src (cellular Src), Fyn, or Yes in immunoblot analysis. E, HUVECs transfected with a scrambled siRNA or siRNA specific for c-Src, Fyn, or Yes were treated with TNFα (10 ng/mL), IL-1β (10 ng/mL), or LPS (500 ng/mL) 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. F, HUVECs transfected with scRNA, c-Src, Fyn, or Yes siRNA were treated with TNFα (10 ng/mL) and IL-1β (10 ng/mL) for 6 h. VCAM1 (vascular cell adhesion molecule 1) and ICAM1 (intercellular adhesion molecule 1) levels were analyzed by immunoblotting, and band intensities were quantified by densitometry, and these data are shown in the bottom. G, HUVECs transfected with scRNA or Fyn siRNA were treated with TNFα (10 ng/mL), IL-1β (10 ng/mL), or LPS (500 ng/mL) overnight, and IL-8 levels in cell supernatants were determined in ELISA. 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.