V5 tag

Polyclonal antibodies (pAb) against E protein and prM protein of ZIKV, E protein of JEV, GP64, and VP39 of baculovirus were used as previously described [16 (link),17 (link),18 (link)]. The pAbs were purified by a Protein A-Sepharose^TM column (Sigma-Aidrich, Darmstadt, Germany). Mouse anti-Flavivirus group antigen antibody (2A10G6) was kindly provided by Dr. Chengfeng Qin (Beijing Institute of Microbiology and Epidemiology, Beijing, China). Mouse antibodies against HA, Strep, and V5-tag were purchased from Sigma (St. Louis, MO, USA). Goat anti-mouse and anti-rabbit IgG-fluorescein isothiocyanate (FITC; Abcam, Cambridge, UK) were used as the secondary antibodies in the immunofluorescence assays (IFA). Thiol-conjugating reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG1332 was kindly provided by Dr. Leike Zhang (Wuhan Institute of Virology, Chinese Academy of Sciences).

Cells were lysed with RIPA buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, 1% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche). Equivalent amounts of proteins determined by the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-C Super; Amersham, Buckinghamshire, UK). Nonspecific antibody binding sites were blocked with skim milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST), then reacted with primary antibodies for HADHα (sc-82185), HADH (sc-55661 and sc-271496) and α Tubulin (sc-5546) from Santa Cruz Biotechnology; actin (NB600-501; Novus Biologicals, Littleton, CO, USA); GAPDH (GTX100118) and TOM70 (GTX85353) from GeneTex (Irvine, CA, USA); Cytochrome C (#556433; BD, Franklin Lakes, New Jersey, USA); Calreticulin (#2891), phospho-STAT1 (Tyr701) (#9171), STAT1 (#9172), phospho-AMPKα (Thr172) (#2535), and AMPKα (#2532) from Cell Signaling Technology(Danvers, MA, USA); GFP (#11814460001; Roche); V5-tag (V8012) and Flag-tag (F7425) from Sigma; or HA-tag (MMS-101R; Covance, Princeton, New Jersey, USA), and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Amersham). Signals were detected by enhanced chemiluminescence (Amersham).