FIGURE 9.

NP transfection and FLNA silencing in an IAV microenvironment were found to activate the JNK stress-signaling pathway while FLNA over-expression in an IAV microenvironment resulted in decreased JNK stress-signaling pathway activation. (A) HEK293 cells were transfected with either pcDNA3.1 empty control plasmid or NP PR8 plasmid in a dose-dependent manner. The cells were harvested with RIPA buffer 24 h post-transfection and 30 μg of protein lysate was loaded onto an SDS-PAGE gel followed by Western blotting. Blots were developed by ECL for FLNA, vinculin (loading control), NP and JNK stress signaling pathway-associated markers. Representative blots are shown from one independent experiment (n = 3) (see Supplementary Figure 9). (B) HEK293 cells were transfected with either control pEGFP-N1 plasmid (3 μg) or FLNA-GFP plasmid (3 μg) followed by IAV PR8 infection (MOI = 5) 24 h post-transfection. The cells were harvested with RIPA buffer 24 h.p.i and 30 μg of protein lysate was loaded onto an SDS-PAGE gel followed by Western blotting. Blots were developed by ECL for JNK stress signaling pathway-associated markers. Representative blots are shown from one independent experiment (n = 3) (see Supplementary Figure 10). See Figure 8 for confirmation of FLNA overexpression and IAV PR8 infection. (C) A549 cells were transfected with either non-targeting control siRNA (200 nM) or FLNA siRNA (200 nM) followed by IAV PR8 infection (MOI = 5) 24 h post-transfection. The cells were harvested with RIPA buffer 24 h.p.i and 30 μg of protein lysate was loaded onto an SDS-PAGE gel followed by Western blotting. Blots were developed by ECL for JNK stress signaling pathway-associated markers. Representative blots are shown from one independent experiment (n = 3) (see Supplementary Figure 11). See Figure 7 for confirmation of FLNA silencing and IAV PR8 infection.