Rabbit anti trim56 pab

Cell lysates were prepared in RIPA buffer and subject to SDS-PAGE and immunoblotting analysis were described previously [25 (link), 43 (link)]. Immunofluorescence staining were performed as previously described [25 (link), 43 (link)]. 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.

To assess whether TRIM56 directly interacts with ZIKV RNA, we first purified MBP-tagged T56-C392 (comprising the C-terminal 392 aa of human TRIM56) protein from E. coli using the pMAL Protein Fusion and Purification System (New England Biolabs, E8000S, USA). To produce a control protein, we purified MBP along with a short stretch of the downstream polylinker (MBP-polylinker) from E. coli transformed with the empty vector pMAL-c4x. ZIKV RNA was extracted using TRIzol from virions in high-titer ZIKV stocks. Subsequently, two micrograms of MBP-polylinker or MBP-T56-C392 protein were incubated with 0.5 μg of ZIKV RNA at room temperature for 30 min, followed by pull-down of MBP-tagged proteins using amylose resin. After three washes, the resins were divided equally into two fractions, which were subjected to RNA isolation by TRIzol and protein extraction by boiling in SDS sample buffer, respectively. Quantification of ZIKV RNA levels was performed by qPCR as described above. MBP-polylinker and MBP-T56-C392 in the protein samples were probed by immunoblotting using rabbit anti-MBP pAb (New England Biolabs) and rabbit anti-TRIM56 pAb (Bethyl Labs).