The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function

Yang, Darong; Li, Nan L.; Wei, Dahai; Liu, Baoming; Guo, Fang; Elbahesh, Husni; Zhang, Yunzhi; Zhou, Zhi; Chen, Guo-Yun; Li, Kui

Cell lysates were prepared in RIPA buffer and subject to SDS-PAGE and immunoblotting analysis were described previously [25, 43] . Immunofluorescence staining were performed as previously described [25, 43] . The following monoclonal (mAb) and polyclonal (pAb) antibodies were utilized: mouse anti-Flag-tag M2 mAb (Sigma); mouse anti-HA-tag mAb (Invivogen); rabbit anti-maltose-binding protein (MBP) pAb (New England Biolabs); mouse anti-flavivirus envelope protein (4G2) mAb (Millipore); mouse anti-ZIKV NS5 mAb, clone 8B8 (Biofront Technologies, kindly provided by Hengli Tang, Florida State University); mouse anti-β-actin mAb (Sigma); rabbit anti-TRIM56 pAb (Bethyl Labs) or rabbit anti-TRIM56 S4091 pAb (generated by immunizing rabbits at Proteintech Group Inc. with a recombinant protein comprising the C-terminal 392 aa of human TRIM56 fused to MBP that was expressed and purified from E. coli); peroxidase-conjugated secondary goat anti-rabbit and goat anti-mouse pAbs (Southern Biotech); FITC-conjugated secondary goat anti-mouse pAb (Southern Biotech). Specifically, FH-T56 was detected by mouse anti-Flag-tag M2 mAb (Sigma); rabbit anti-TRIM56 S4091 pAb was used to detect endogenous T56 protein in HeLa cell lysates; and rabbit anti-TRIM56 pAb (Bethyl Labs) was used for other T56 immunoblotting experiments.