Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses

Figure 2

(a) Influence of 100 μg/ml binase at 12 h p.i. on proteome of A549 cells. Two-dimensional difference gel electrophoresis (2D-DIGE) of proteins isolated from nontreated ((i), green labeling by Cy3) and binase-treated cells ((ii), red labeling by Cy5); (iii) merged image, unchanged protein spots appeared yellow. High resolution 2D-DIGE images were generated using Typhoon FLA 9500 Imager; samples were separated using narrow range of IEF tube gel, pH 3–8; gels cover 15–150 kDa range. (b) Real-time RT-PCR analysis of mRNA transcripts of housekeeping genes (beta-actin, ACTB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH) in noninfected, binase-treated (100 μg/ml, 12 h), and nontreated A549 cells. (c) H1N1pdm viral NP mRNA in infected nontreated A549 cells (1) and treated by 100 μg/ml binase after 12 h (2). GAPDH mRNA was used as a loading control to normalize viral mRNA accumulation. 10⁶ cells from each sample were used to measure mRNA levels using Roche Light Cycler 480 system and target-specific primers. Statistical significance was calculated using GraphPad Prism 5.0 Software and assessed by Student’s t-test: .

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