Ab179434

The cell lysates were prepared, and 10 µg of TRAF6 (Santa Cruz Biotechnology # sc‐8409) antibodies and 1 mg of each protein sample were used for co‐IP assays (Thermo Scientific, USA) to detect K48‐specific (1:1000, Abcam # ab140601) and K63‐specific (1:1000, Abcam # ab179434) polyubiquitination. The eluted protein samples analysed by Western blot.

The coding region of human USP2A (accession number NM_004205) without the stop codon was cloned into pcDNA3.2/V5-DEST (Thermo Fisher Scientific) using BP clonase II (Thermo Fisher Scientific). The resultant plasmid encoded C-terminal V5-tagged USP2A. Halo-tagged Oct-1 and Oct-2 expression plasmids were purchased from Promega (Madison, WI). The HA-tagged ubiquitin plasmid was purchased from Addgene (Cambridge, MA). The USP2A, or control pcDNA3.2/V5-DEST plasmid, plasmids encoding Halo-tagged Oct-1 or Oct-2 and HA-tagged ubiquitin were transfected into HEK293FT cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). The pull-down and elution of Oct proteins were performed using Magne HaloTag beads (Promega) and HaloTEV protease (Promega) as per the manufacturer's instructions. For detection of Western blot bands, corresponding to K48- and K63-linked ubiquitin chains, Oct proteins, HA-tagged ubiquitin, and V5-tagged USP2, 1000-fold-diluted antibodies against polyubiquitin chains formed by K48 residues (ab140601; Abcam) and K63 residues (ab179434; Abcam), Oct-1 (A301-717A; Bethyl Laboratories), Oct-2 (ab179808; Abcam), HA-tag (#3724; CST), and V5-tag (A190-120F; Bethyl Laboratories) were used as the primary antibodies.

Immunoprecipitation of TRAF6 was performed using an anti-TRAF6 (ab33915; Abcam) antibody with a Pierce Crosslink IP Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. For detection of Western blot bands, TRAF6 (ab33915; Abcam), polyubiquitin chains formed by K63-linked (ab179434; Abcam) and K48-linked (ab140601; Abcam) primary antibodies were used (1000-fold dilution).

Viral proteins were detected using in-house produced rat polyclonal antibodies against NP protein (1:5000; [24 (link)]) and against NS1 protein [25 (link)]. Cellular proteins and their modifications were detected using commercially available antibodies as follows: rabbit polyclonal antibodies to H3K79me2 (D15E8), H3 (D1H2), and CHD1a (D8C2) (1:1000) from Cell Signaling (Danvers, MA, USA); rabbit polyclonal antibodies anti-TRIM25 (EPR7315) (ab167154), anti-RIG-I (DDX58) (ab45428), and anti-Ubiquitin UbK63 (linkage-specific K63) (EPR8590-448) (ab179434) from Abcam (Cambridge, United Kingdom); mouse monoclonal antibodies anti-MAVS (E-3 (sc-166583); 1:1000), and anti-β-tubulin (1:1000) from Santa Cruz (Dallas, TX, USA) and Sigma-Aldrich, respectively.

Cells or testis cryosections were fixed with 4 % paraformaldehyde in PBS for 1 h at room temperature (RT). Followed by permeabilization with 0.025% Triton X-100, samples were incubated in 5 % bovine serum albumin (BSA) for 1 h at RT and then incubated with primary antibody diluted in BSA overnight at 4 °C. The next day, samples were washed with PBS 3 times (10 min per wash), followed by incubation with Alexa Fluor 488/594 secondary antibodies at RT for 1 to 2 h, and 4,6-diamidino-2-phenylindole was used to visualize the nuclei. Immunofluorescent (IF) images were obtained using a confocal microscope (LSM 900, Zeiss, Germany). The primary antibodies used for IF are summarized as follows: SERBP1 (Abnova, D5C5Z), G3BP1 (Proteintech, 66486-1-AP), TIAR (Proteintech, 66907-1-Ig), PABPC1 (Abcam, Ab21060), 20S (Abcam, ab22674), VCP (Proteintech, 60316-1-lg), FAF2 (Proteintech, 16251-1-AP), ubiquitin (Abcam, Ab7780), K63 ubiquitin (Abcam, Ab179434), and K48 ubiquitin (Abcam, Ab140601).