Figure 4

RSV gene expression in profilin-depleted cells. Panels A, B: siRNA-mediated knock down of profilin. A549 monolayer was transfected with 0, 5, 50, or 200 nM profilin siRNA, and then infected with RSV. Lane U indicates control cells that were neither transfected nor infected. About 40 μg of total cellular protein was subjected to immunoblot in each lane using antibody against profilin (Pr) (panel A) or RSV P (panel B). The chemiluminographs are presented. Note that profilin siRNA did not appreciably affect the P protein. Panel C, D: Immunoblot of RSV proteins. A549 (panel C) or L2 (panel D) cells were pre-treated with 0, 200, or 400 nM profilin siRNA and then infected with RSV as in the previous panels. A total of 40 μg of total protein from the infected cells were subjected to immunoblot using an antibody mixture against RSV G, N, P, and M proteins. Lane U represents identical amount of protein from the respective uninfected cells. All bands were digitally scanned by a Fujifilm LAS-1000 Intelligent Dark Box II Image Reader (Fujifilm, Edison, NJ), and the intensities integrated and added using the Image Gauge v4.0 software. Total intensities of the G, P, N, and M bands of the siRNA-treated lanes, expressed as percent of the untreated (0 nM) lane, are presented at the bottom. Values represent mean of three experiments with the range in ±. In panels A-D, the protein size markers are indicated on the left in kDa. Panel E: RT-PCR quantitation of N and F mRNA and genomic RNA of RSV. Design of primers and the procedure of RT-PCR have been described [25]. All values were mean of three experiments with the range indicated by bars. Results were normalized against control actin RNA amplified by RT-PCR using primers described previously [25].