Journal: eLife
Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7
doi: 10.7554/eLife.102277
Figure Lengend Snippet: ( A ) The nonstructural proteins nsp8, nsp11, and nsp16 could be restored by the proteasome inhibitor MG132. HEK293T cells in 12-well plates were transfected with the plasmids of 16 nonstructural proteins (nsp1–16) encoded by SARS-CoV-2. Thirty-six hours later, the cells were treated with MG132 (10 µM) or DMSO for 12 hr before collection. The protein level was detected by immunoblotting (IB). Quantification of nsp protein levels relative to the control protein is shown. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: *p<0.05; **p<0.01; ***p<0.001. ( B ) Proteasomal inhibitors but no other inhibitors stabilized nsp16 protein. HEK293T cells transfected with the nsp16-Flag expression vector were treated with dimethyl sulfoxide (DMSO), MG132 (10 µM), Bortezomib (10 µM), Carfilzomib (10 µM), Bafilomycin A1 (5 µM), Vinblastine (2.5 µM), or NH 4 Cl (2.5 µM) for 12 hr prior to harvest. The cell lysates were analyzed by anti-Flag antibody. ( C, D ) The half-life of nsp16 was prolonged by the proteasome inhibitor MG132. (C) HEK293T cells were transfected with the nsp16-Flag-expressing plasmids. 12 hr later, the cells were treated with DMSO or MG132 (10 µM) for 12 hr, then 50 µg/ml cycloheximide (CHX) was added. Cells were harvested at the indicated times to detect the level of viral protein by anti-Flag antibody. (D) Quantification of nsp16 protein levels relative to tubulin at different time points is shown. The half-life of the nsp16 protein was determined based on protein quantification using ImageJ, combined with the protein half-life formula for calculation. Results are shown as mean ± SD (n = 3 independent experiments). ***p<0.001 by a two-tailed t-test. ( E ) Samples were prepared for mass spectrometry, and nsp16 interacting proteins were obtained by immunoprecipitation (IP) (created with BioRender.com and the agreement no. is XR281XWMTN). The plasmids were transfected into HEK293T cells for 48 hr. Treat cells with or without MG132 (10 µM) for 12 hr prior to harvest. The whole-cell lysates were incubated with protein G agarose beads conjugated with anti-Flag antibodies and used for IB with anti-Flag antibodies to detect the nsp16 protein. Samples enriched for proteins were analyzed by mass spectrometry. Figure 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 1—source data 2. Original files for western blot analysis displayed in . Figure 1—source data 3. Numerical data obtained during experiments represented in .
Article Snippet: The drugs used in this study were as follows: MG132 (catalog no. S2619), Bafilomycin A1 (catalog no. S1413), Bortezomib (catalog no. S1013), Carfilzomib (catalog no. S2853), and Vinblastine (catalog no. S4504) were purchased from Selleck (Houston, TX, USA).
Techniques: Transfection, Western Blot, Control, Two Tailed Test, Expressing, Plasmid Preparation, Mass Spectrometry, Immunoprecipitation, Incubation
