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. 2020 Jul 1;10:10755. doi: 10.1038/s41598-020-67549-7

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© The Author(s) 2020

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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Ras activation was decreased in the RasGRP1 knocked down cell lines treated with LPS. (A) BV2 cells were infected with lentiviral particles containing scrambled or shRasGRP1 RNA. Infected cells were selected by puromycin treatment (5 μg/ml). The expression level of RasGRP1 was analyzed by western blot. (B) BV2 cells expressing scrambled or shRasGRP1 RNA were treated with LPS (1 μg/ml) for 40 min. The expression level of the downstream signal cascade of RasGRP1 was analyzed by western blot. (C, D) BV2 cells expressing scrambled or shRasGRP1 RNA were treated with LPS (1 μg/ml) for 20 min. (C) GST-fused Ras binding domain (RBD) was used for the pulldown assay to determine the level of GTP-bound active Ras protein in the cell lysates. A parallel western blot analysis of the total cell lysate was performed to determine the total Ras expression level. (D) Data were quantitatively analyzed by densitometry. These experiments were repeated three times. Statistical analysis was performed using Student's t-test (mean ± SD; n = 3; **P < 0.01, ***P < 0.005).