Decanoyl rvkr cmk

Embryos were raised for 24 h after injection with the EMMA–Mmp11a construct and heat shocked as described above, but with the addition of 50 nM furin inhibitor I (Decanoyl-RVKR-CMK, catalogue# 3501/1; R&D Systems, Minneapolis, MD, USA), or vehicle (0.1% DMSO) to the ERM in which they were incubated.

Anti-HA-Tag antibody (sc-7392; Santa Cruz, Dallas, TX) was used to detect TMPRSS2 and anti-c-Myc antibody (sc-40, Santa Cruz) was used to detect furin, while anti-ACE2 (10108-T60; Sino Biological Inc., Beijing, China) and anti-CTSL (10486-RP02 Sino Biological Inc.) antibodies were used to detect ACE2 and CTSL, respectively. Anti-S2 antibody against SARS-CoV-2 S protein was generated in-house by immunizing mice with recombinant S2 protein. The anti-VSV M protein antibody was purchased from KeraFast (EB0011; Boston, MA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (CW0102S; CWbiotech, Beijing, China) and HRP-conjugated goat anti-rabbit IgG (SSA004; Sino Biological Inc.) were used as secondary antibodies. Nine monoclonal antibodies against SARS-CoV-2 S protein were used in the neutralization assays, of which CB6 and CA1 were gifts from Dr. Jinghua Yan of University of Chinese Academy of Sciences [51 (link)]; X593 was from Dr. X. Sunney Xie of Peking University [52 (link)]; H014 and H00S002 were from Dr. Liangzhi Xie of Sino Biological Company [53 (link)]; 1F11, A261–262, A157, and A247 were from Dr. Linqi Zhang of Tsinghua University [54 (link)]. Furin inhibitor (Decanoyl-RVKR-CMK) was from R&D Systems (Bristol, UK, Cat#: 3501) Cathepsin inhibitor (E64D) was from Apexbio (Shanghai, China, Cat#: A1903).

The reduction of luciferase gene expression was detected to evaluate the infection inhibition effect of hACE2 protein (expressed in HEK293 cells, Sino Biological), protease inhibitor Decanoyl-RVKR-CMK (Furin inhibitor, R&D Systems), protease inhibitor Camostat (TMPRSS2 inhibitor, Tocris Bioscience), protease inhibitor E64D (Cathepsin L inhibitor, APExBIO), monoclonal antibody, and sera. As described previously^{19 (link),20 (link)}, the tested samples were diluted (starting with 30 times dilution and three times gradient, a total of eight gradients), after which the virus solution was added. On each 96-well plate, eight virus control wells and eight cell control wells were arranged. In the virus control wells, no test sample but only virus solution was added; in the cell control wells, no virus solution but only a complete culture medium was added. The 96-well plates were incubated at 37 °C for 1 h, after which trypsinized Huh7 cells (2 × 10⁴/100 μL) were added to each well. After incubation for 24 h in an atmosphere comprising 5% CO₂ at 37 °C, luminescence was measured as described above. The EC₅₀ value of each sample was calculated using the Reed–Muench method.