FIG 2.

RSV infection promotes NLRC5 expression. (A) Gene induction determined by RT-PCR. A549 cells were infected with RSV at different MOIs (0.1, 0.3, and 1.0) for 24 h. Total RNA was isolated for the detection of NLRC5, IFN-β, RIG-I, and STAT1 by RT-PCR. GAPDH expression was used as an internal control. (B) Demonstration of a monoclonal antibody that recognizes both overexpressed and IFN-γ-induced NLRC5. A recombinant protein corresponding to the C-terminal 258 amino acid residues of human NLRC5 was prepared in E. coli and used to immunize BALB/c mice to generate a monoclonal antibody that could be used for immunoblotting (IB) studies. Clone 7 recognized two protein bands at approximately 230 kDa from p3x-FLAG-NLRC5-transfected 293T cells. It also recognized a pair of protein bands at approximately 230 kDa from IFN-γ-treated A549 cells. A band at approximately 130 kDa was a nonspecific band (ns). (C) Dose-response and time course studies of NLRC5 induction by RSV infection. A549 cells were infected with RSV at various MOIs, as indicated, for 24 h or at an MOI of 1 for the indicated times. NLRC5 induction was detected using clone 7. RSV F protein expression was used as an indication of infection, while GAPDH was used as a loading control. (D) Pretreatment of A549 cells with siNLRC5 suppresses NLRC5 induction. A549 cells were left untreated or treated with two different siRNAs targeting NLRC5 (siNLRC5 number 1 and number 2) or an SC. The cells were then infected with RSV at an MOI of 1 and used for detection of NLRC5 induction and F protein expression. GAPDH expression was used as a loading control.