Figure 4. Simultaneously knockdown both Raptor and Rictor block viral protein synthesis. Lenti-viral short- hairpin RNAs (shRNA) were used to knockdown expression of both Raptor and Rictor in A549 cells. Double shRaptor/shRictor-A549 cells were infected with RSV NH409A. Cellular and viral protein expression was measured at 24 h post infection (m.o.i = 0.2). Asterisk ( * ) or non-significant (ns) is compared to Scramble/RSV control. ( A ) Western blot analysis of cellular and protein expression. ( B – H ) Quantification of protein synthesis displayed in panel A (n = 3). ( B ) Raptor/β-actin; ( C ) phospho-S6K1/S6K1; ( D ) total ribosomal protein S6/β- actin; ( E ) Rictor/β-actin; ( F ) phospho-Akt/Akt; ( G ) RSV nucleoprotein N/β-actin; ( H ) RSV fusion protein F1/β- actin. Error bars, SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s.