human plvx puro trim21 gfp  (Addgene inc)

Journal: PLOS Pathogens

Article Title: SARS-CoV-2 Nsp1 suppresses the canonical NF-κB pathway by promoting ubiquitin-dependent degradation of TAK1 kinase

doi: 10.1371/journal.ppat.1014191

Figure Lengend Snippet: a : HEK293T cells were transfected with plasmids expressing FLAG-tagged TAK1 (TAK1-FLAG) and wild-type (WT) or mutant (K48R/K63R) HA-Ub, and 24 h later, were transfected again with a plasmid expressing cMYC-tagged TRIM21 (cMYC-TRIM21) or GFP (GFP-cMYC, Ctrl). Ubiquitinated TAK1 was immunoprecipitated (IP) by anti-FLAG magnetic beads, and the ubiquitination level was analyzed by an anti-HA antibody. GFP (green arrowhead) and TRIM21 (red arrowhead) were detected by anti-cMYC and anti-TAK1 with anti-FLAG antibodies in a-b . The relative signals of HA-Ub and TAK1-FLAG quantified by ImageJ are shown in the right panels in a & e . b : Plasmids expressing cMYC-tagged full-length (FL) or truncated TRIM21 mutants expressing only SPRY or lacking (Δ) the SPRY domain were constructed (left panel). SPRY, SPla and the RYanodine Receptor domain. HEK293T cells were cotransfected with plasmids expressing TAK1-FLAG and FL or truncated cMYC-TRIM21 or GFP. Immunoprecipitation (IP) was performed using anti-cMYC magnetic beads (right panel). c : HEK293T cells were cotransfected with plasmids expressing cMYC-TRIM21 and FLAG-tagged full-length (FL) or truncated N-terminal (1-303 aa, N) or C-terminal (304-606 aa, C) TAK1 mutants. FLAG-tagged GFP (green arrowhead) and TAK1 (blue arrowhead) were detected with an anti-FLAG antibody. d : HEK293T cells were transfected with plasmids expressing HA-tagged TAK1 and cMYC-TRIM21 24 h later and transfected with a plasmid expressing FLAG-tagged Nsp1 (red arrowheads) or a GFP control (Ctrl, green arrowhead). Immunoprecipitation (IP) was performed using anti-HA magnetic beads. The relative signals of cMYC-TRIM21 and TAK1-HA quantified using ImageJ are shown in the right panel. e : HEK293T cells were transfected with a lentiviral vector encoding shRNA targeting TRIM21 (shTRIM21) or luciferase (shLuc) as a control. After puromycin (10 μg/mL) selection for 72 hr, the cells were transfected with an HA-Ub plasmid, and 24 hr later, transfected with an Nsp-cMYC (red arrowheads) or a GFP-cMYC plasmid (Ctrl, green arrowheads). The results in a , d and e are representative of two independent experiments.

Article Snippet: Nsp1 binding kinetics against TAK1 (SinoBiological, #M15-13G) or TRIM21 (MCE, #HY- P71791 ) were measured by surface plasmon resonance (SPR) on a BIAcore 8K+ (Cytiva).

Techniques: Transfection, Expressing, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Ubiquitin Proteomics, Construct, Control, shRNA, Luciferase, Selection

Journal: PLOS Pathogens

Article Title: SARS-CoV-2 Nsp1 suppresses the canonical NF-κB pathway by promoting ubiquitin-dependent degradation of TAK1 kinase

doi: 10.1371/journal.ppat.1014191

Figure Lengend Snippet: In uninfected cells (left), TAB1 binds to TAK1, and the TAK1-TAB1 complex phosphorylates downstream regulators, including IκBα, p38, and Erk, which in turn activate the downstream NF-κB, MAPK and AP-1 pathways. In SARS-CoV-2-infected cells (right), Nsp1 binds to TAK1 at the N-terminal TAB1-binding domain, preventing the formation of the TAK1-TAB1 complex and promoting the binding of TRIM21 to the C-terminus of TAK1. As a result, Nsp1 promotes the proteasomal degradation of TAK1 by enhancing the TRIM21-dependent K48-linked polyubiquitination (Ub) of TAK1, thereby attenuating the activity of the NF-κB and AP-1 pathways. Created in BioRender. Yang, Q. (2026) https://BioRender.com/41xkykd .

Article Snippet: Nsp1 binding kinetics against TAK1 (SinoBiological, #M15-13G) or TRIM21 (MCE, #HY- P71791 ) were measured by surface plasmon resonance (SPR) on a BIAcore 8K+ (Cytiva).

Techniques: Infection, Binding Assay, Activity Assay