DNA constructs pHA‐RelB and pQC‐RelB were described previously [26 ]. Maxine L. Linial (Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA) provided the pLTR‐luc [28 ], pIP‐luc [28 ], and pcPFV (PFV full‐length infectious clone) [29 ]. DNA clones PFV p3.1‐Tas and pFlag‐Tas were generated by inserting the region from nucleotide 9434 to nucleotide 10336 encompassing the orf1 into the pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) or pCMV‐Tag2B (Stratagene, La Jolla, CA, USA) vector.
Rabbit anti‐RelB antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti‐Flag (M2) and mouse anti‐HA antibodies were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Mouse anti‐tubulin, rabbit anti‐HA, and horseradish peroxidase‐conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein‐conjugated anti‐mouse and anti‐rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). DAPI (4′,6‐diamidino‐2‐phenylindole) and protein A beads were purchased from Sigma‐Aldrich. Antibodies against PFV Gag and PFV Tas were generated in BALB/c mice using bacterially purified PFV Gag (180‐433aa) and PFV Tas protein as immunogens. Because PFV Bet protein is a fusion protein produced by 88 amino acids at the 5' end of Tas and the complete Bel2 coding protein, this Tas antibody can be used to detect Bet protein. These polyclonal anti‐serums were used for western blotting and immunofluorescence imaging.
Rabbit anti‐RelB antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti‐Flag (M2) and mouse anti‐HA antibodies were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Mouse anti‐tubulin, rabbit anti‐HA, and horseradish peroxidase‐conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein‐conjugated anti‐mouse and anti‐rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). DAPI (4′,6‐diamidino‐2‐phenylindole) and protein A beads were purchased from Sigma‐Aldrich. Antibodies against PFV Gag and PFV Tas were generated in BALB/c mice using bacterially purified PFV Gag (180‐433aa) and PFV Tas protein as immunogens. Because PFV Bet protein is a fusion protein produced by 88 amino acids at the 5' end of Tas and the complete Bel2 coding protein, this Tas antibody can be used to detect Bet protein. These polyclonal anti‐serums were used for western blotting and immunofluorescence imaging.
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