HEK-293T cells transfected with pHA-Ubiquitination, pEGFP-METTL14, or pMYC-HRD1 were lysed in NP-40 buffer after 48 hpt for Co-IP assay. Cell lysates were processed for western blotting using rabbit anti-METTL14/MYC antibody (Proteintech, China), rabbit anti-HA antibody (Sigma, USA), and rabbit anti-β-actin antibody (ABclonal, China). Additionally, to examine effect of NS5B on HRD1 E3 ligase activity, HEK-293T cells transfected with pHA-Ubiquitination, pEGFP-METTL14, pFlag-NS5B, or pMYC-HRD1 were lysed in NP-40 buffer after 48 hpt for Co-IP assay. Cell lysates were processed for western blotting using rabbit anti-METTL14/MYC antibody (Proteintech, China), rabbit anti-HA antibody (Sigma, USA), and rabbit anti-β-actin antibody (ABclonal, China). For studying polyubiquitination of METTL14, cells transfected with pHis-Ub (WT and -K6, -K11, -K27, -K29, or -K63), pEGFP- METTL14, and pHA-HRD1. At 48 hpt, cells were lysed in NP-40 lysis buffer for the same experiment operation as above. Cell lysates were processed for western blotting using rabbit anti- METTL14/His antibody (Proteintech, China), rabbit anti-HA antibody, and rabbit anti-β-actin antibody.
Chen J., Song H.X., Hu J.H., Bai J.S., Li X.H., Sun R.C., Zhao B.Q., Li M.Z, & Zhou B. (2024). Classical swine fever virus non-structural protein 5B hijacks host METTL14-mediated m6A modification to counteract host antiviral immune response. PLOS Pathogens, 20(3), e1012130.
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