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. 2019 Feb 14;9:2096. doi: 10.1038/s41598-018-38312-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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SubA fails to mediate thrombin-induced inflammatory response. Confluent HPAEC were biotinylated using Sulfo-NHS-SS-Biotin, subsequently lysed and labeled cell surface proteins were isolated using streptavidin beads and then separated on an SDS-PAGE and probed for BiP, VE-cadherin and GAPDH (A). TL is total lysate. HPAEC were treated with 0.1 µg/ml of SubA and SubAB for the indicated time points. Cell lysates and corresponding cell supernatants were immunoblotted for BiP/GRP78 to monitor the cleavage of BiP/GRP78 by SubA and SubAB (B). The blot in 4B is cropped from two different parts of the same gel, as shown by white space. HPAEC were transfected with NF-κBLUC and pTKRLUC constructs by DEAE-dextran as described in Materials and Methods section. Following transfection cells were treated with 0.1 µg/ml of SubA for 3 h and then by 5 U/ml of thrombin for 6 h. Total cell lysates were prepared and assayed for Firefly and Renilla luciferase activities (C). Confluent HPAEC were pretreated with SubA for 3 h followed by thrombin treatment (5 U/ml) for 6 h. Cell lysates were probed for ICAM-1(D) and cell supernatants were analyzed for IL-8 (E) and IL-6 (F) by ELISA. HPAEC were pretreated with SubAB for 3 h followed by thrombin treatment (5U/ml) for 6 h. Total cell lysates were probed for IL-6 (G) by ELISA.