Anti SARS 3Cl Protease  

Western blot using Rockland's Protein A Purified anti-SARS CoV 3CL Protease antibody shows detection of a 34-kDa band corresponding to the protein. Approx. 100 ng of protein was loaded for SDS-PAGE and transferred onto nitrocellulose. The blot was incubated with a 1:5,000 dilution of the antibody at room temperature for 1 h followed by detection using IRDye^TM800 labeled Goat-a-Rabbit IgG [H&L] (611-132-122) diluted 1:10,000. The fluorescence image was captured using the Odyssey^(R) Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

The luciferase complementation assay accurately reflects concentration-dependent inhibition of SARS-CoV-2 3CLpro-mediated cleavage. (A) Western blotting to examine reporter cleavage. 293T cells were transfected with the lentiviral vector containing 3CLpro？WT and the S-L-GFP reporter cassette, and DMSO (control) or GC376 (0.1, 0.4, 1.6, 6.3, 25, 100, or 200 μM) was added. Mock transfection (MT) and 3CLpro？C145A S-L-GFP were included as controls. On the GFP and LgBiT (L) panels, the bottom band corresponds to cleaved reporter (L-GFP, 46 kDa, indicated by arrows), while the top band corresponds to uncleaved reporter (S-L-GFP, 49 kDa). (B) Quantification of the percentage of uncleaved reporter from three independent western blots based on GFP (circle) or L (triangle) detection. (C) NanoBiT luciferase activity and cell viability. 293T cells were transfected with the S-L-GFP lentiviral vector, and DMSO (control) or GC376 (0.1, 0.4, 1.6, 6.3, 25, or 100 μM) was added. NanoBiT activity (circle, left axis) and cell viability (measured with the CellTiter-Glo 2.0 assay; triangle, right axis) were analyzed 30 h post-transfection and are expressed as relative luciferase units (RLU). Cell viability data were normalized to the DMSO sample, which was set to 100%. NanoBiT data were normalized to the DMSO and 100 μM GC376 samples, which were set to 0 and 100%, respectively. The data represent the mean ± standard deviation of four independent experiments; ns: not significant; *？p？< 0.05, **？p？< 0.01, ***？p？< 0.001 (relative to no-drug; one-way ANOVA with Dunnett’s post-test). Fig 2. PMID:？33498923

(C) SDS-PAGE, Edman sequencing (green) and immunoblot validation of human MAP4K5 and CREB1 substrates incubated with 3CLpro+/− inhibitor GC376, or 3CLpro-C145A (1:5 mol/mol, E:S). ΔMAP4K5 or ΔCREB1, no sequence obtained. Fig 4. PMID:？34672947