Author: Ting Gao; Mingdong Hu; Xiaopeng Zhang; Hongzhen Li; Lin Zhu; Hainan Liu; Qincai Dong; Zhang Zhang; Zhongyi Wang; Yong Hu; Yangbo Fu; Yanwen Jin; Kaitong Li; Songtao Zhao; Yongjiu Xiao; Shuping Luo; Lufeng Li; Lingfang Zhao; Junli Liu; Huailong Zhao; Yue Liu; Weihong Yang; Jing Peng; Xiaoyu Chen; Ping Li; Yaoning Liu; Yonghong Xie; Jibo Song; Lu Zhang; Qingjun Ma; Xiuwu Bian; Wei Chen; Xuan Liu; Qing Mao; Cheng Cao
Title: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation Document date: 2020_3_30
ID: dxs8ggyh_35
Snippet: MASP-2 auto-activation and C4 cleavage assay 15 Purified MASP-2 (8 nM) was incubated at 37°C in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 2 mM CaCl 2 with purified C4 (Calbiochem), recombined MBL (Calbiochem), mannan (Sigma), and SARS-CoV N protein at concentrations of 50 nM, 30 nM, 15 ng/ml, and 10 nM, respectively. The cleavage was followed by SDS-PAGE under reducing conditions, and the C4alpha' fragments, which are left by C4a release after C.....
Document: MASP-2 auto-activation and C4 cleavage assay 15 Purified MASP-2 (8 nM) was incubated at 37°C in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 2 mM CaCl 2 with purified C4 (Calbiochem), recombined MBL (Calbiochem), mannan (Sigma), and SARS-CoV N protein at concentrations of 50 nM, 30 nM, 15 ng/ml, and 10 nM, respectively. The cleavage was followed by SDS-PAGE under reducing conditions, and the C4alpha' fragments, which are left by C4a release after C4 cleavage and can indicate C4 20 activation, and MASP-2 were detected by immunoblot analysis with anti-C4alpha chain antibody (Santa Cruz) or anti-Flag antibody (Sigma). C4b containing C4alpha' but not C4a was used to indicate C4alpha' band position.
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