FIGURE 6.

p38MAPK plays a role in the nucleocytoplasmic export of NP. A, top, MDCK cells were treated continuously with different concentrations (10–20 μm) of SB203580, SB202474, or DMSO (controls) 1 h before PR8 infection (I) (1.5 MOI). At postinfection hour 8, cells were collected, fixed, labeled with anti-NP Abs (green fluorescence), subjected to nuclear DAPI staining (blue staining), and analyzed by confocal microscopy. Merged images of the infected cells are shown in the figure. Results are shown for one representative experiments of two performed. Bottom, equal numbers of MDCK cells were treated as described before. At postinfection hour 8, cytosolic and nuclear extracts were immunoprecipitated (IP) with anti-NP Abs, subjected to SDS-PAGE and immunoblotted with anti-influenza virus Abs. PIP, postimmunoprecipitation supernatants. Densitometric analysis of NP expression is shown on the right. Results are expressed as mean ratio of nuclear NP to cytosolic NP ± S.D. from two separate experiments. B, left, MDCK cells were transfected with siRNA against p38MAPK (si p38) or with scrambled siRNA (si Scr), and 12 h later the transfected cells were infected with PR8 (1.5 MOI). Eight hours postinfection (21 h post-transfection, when down-regulation of p38MAPK expression in si p38 cells peaked), cells were collected, fixed, labeled with anti-NP Abs (NP), subjected to nuclear DAPI staining (Dapi), and analyzed by confocal microscopy. Merged images are shown for each condition in the third column. Results are shown for one representative experiment of two performed. Right, si p38 and si Scr cells were infected with PR8, and 8 h after infection (21 h post-transfection), cytosolic and nuclear extracts were subjected to SDS-PAGE and immunoblotted with anti-influenza A virus Abs. Bottom, densitometric analysis of NP expression. Results are expressed as mean ratio of cytosolic NP to α-tubulin ± S.D. and nuclear NP to Sp1 ± S.D. from two separate experiments.