Polyinosinic polycytidylic poly 1 c

The nsp11 coding sequence was PCR-amplified from the FL12 strain of PRRSV and was inserted into the retroviral expressing vector pLNCX2 (Clontech) and mammalian expression vector pXJ41 with a FLAG tag at its N-terminus using the following primers: forward 5′-AAACTCGAGGCCACCATGGGGTCGAGCTCCCCGCTCCC-3′ and reverse 5′-GCGGCCGCTTACTTATCGTCGTCATCCTTGTAATCTTCAAGTTGAAAATAGGC-3′. The translation initiation and termination codons were added to the nsp11 coding sequence. The anti-FLAG monoclonal antibody (MAb M2, Sigma) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) is a synthetic nucleoside that is an analog of thymidine and is commonly used in the detection of proliferating cells. Polyinosinic:polycytidylic (poly [I:C]) as a double-stranded RNA analog was purchased from Sigma. A donkey anti-rabbit antibody conjugated with Texas Red and a goat anti-mouse antibody conjugated with FITC were purchased from Invitrogen (Carlsbad, CA, USA). The nsp11-specific rabbit antibody was generated in our laboratory using recombinant proteins described as follows.

Antibodies and chemicals used in the present study are listed as follows. Anti (α)-PRRSV-nsp1β PAb (rabbit polyclonal antibody) specific for PRRSV-2 nsp1β was generated at the Immunological Research Center, University of Illinois at Urbana-Champaign (Urbana, IL). α-N protein MAb (MR40; mouse monoclonal antibody) was obtained from E. Nelson (South Dakota State University, Brookings, SD). α-p65 Mab (mouse) (F-6, sc-8008) was purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). Alexa-Flour 488-conjugated and Alexa-Flour 568-conjugated secondary antibodies were obtained from ThermoFisher (Rockford, IL). Human tumor necrosis factor-α (TNF-α) (8902) was purchased from Cell Signaling (Danvers, MA). DAPI (4′, 6′-diamidino-2-phenylindol) and Polyinosinic:polycytidylic [poly (I:C)] were obtained from Sigma (St. Louis, MO). Human and porcine recombinant IFN-β were purchased from Calbiochem (San Diego, CA), and for stimulation, 1000 unit/ml was added to cells for 6 h.