Fig 1: Both TRIM13 and RNF121 are required for IL-8, IP-10, IL-1ß, and IL-6 induction in SARS-CoV-2-infected cells. (A) HEK293T-ACE2, HEK293T-TAK1-KO-ACE2, or HEK293T-NEMO-KO-ACE2 cells were infected with SARS-CoV-2. At 24 h after infection, the level of IL-8 and IP-10 was determined by RT-qPCR. The indicated mRNA levels were normalized to GAPDH expression. (B to D) HEK293T-ACE2 cells were transduced with the indicated lentiviral vector expressing shRNA. At 48 h after transduction, cells were infected with SARS-CoV-2. At 24 h after infection, the levels of TRIM13, RNF121 (B), IL-8, IP-10, IL-1ß, IL-6 (C), and SARS-CoV-2 RNA (D) were determined by RT-qPCR. The indicated mRNA levels were normalized to GAPDH expression. Results are shown as the mean ± SD of three independent experiments. **, P < 0.01 (Student’s t test).
Fig 2: Importance of ORF7a ubiquitination by RNF121 for NF-κB activation. (A) HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-ORF7a, or pcDNA3.1-HA-ORF7a-K119R, along with pGL4-NF-κB-LucP2 and pNull-RLuc. The level of luciferase activity was determined at 24 h posttransfection. The firefly luciferase activity was normalized to Renilla luciferase activity. An empty plasmid was used as a control and set to 1. Results are shown as the mean ± SD of three independent experiments. **, P < 0.01 (Student’s t test). (B) HEK293T cells were transduced with the indicated lentiviral vector expressing shRNA. At 48 h postransduction, cells were transfected with the indicated plasmids. Cells were lysed with 1× IP lysis buffer at 24 h posttransfection, followed by ubiquitination assay. The numerical values below the blots indicate the levels of ubiquitinated ORF7a. (C) HEK293T cells were transfected with the indicated plasmids. At 48 h posttransfection, cells were lysed with IP lysis buffer, followed by IP using anti-HA antibody. The immunocomplex was subjected to Western blotting using the indicated antibodies. (D to F) HEK293T or RNF121-knockdown 293T cells were transfected with the indicated plasmid. At 48 h posttransfection, cells were subjected to Western blotting (D), NF-κB reporter assay (E), or RT-qPCR (F). Results are shown as the mean ± SD of three independent experiments. **, P < 0.01 (Student’s t test). (G) HEK293T cells or RNF121-knockdown HEK293T cells were transfected with the indicated plasmids. At 48 h posttransfection, cells were lysed with IP lysis buffer, followed by IP using anti-HA antibody. The immunocomplex was subjected to Western blotting using the indicated antibodies. (H) HEK293T cells were transfected with the indicated plasmids. At 48 h posttransfection, cells were lysed with IP lysis buffer, followed by IP using anti-HA antibody. The immunocomplex was subjected to Western blotting using the indicated antibodies.
Supplier Page from MilliporeSigma for Anti-RNF121 antibody produced in rabbit