Activation of PKR Inhibits Cell Migration Figure 5. Effect of p38 MAPK inhibitor on PIC-stimulated cell signaling. MDA-MB231 cells were pretreated with a selective p38 MAPK inhibitor (SB203580, 5 mM) for 2 hours followed by PIC (0 or 10 ng/ml) treatments for an additional 6 hours. A: After the treatment, cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin. The expression of actin served as a loading control. B: The relative levels of pPKR, pp38, pMK2, pLIMK and pcofilin were quantified as described under the Materials and Methods and normalized to the expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin respectively. * denotes a significant difference from untreated controls. C: The expression of phospho-cofilin and phospho-MK2 was visualized by immunofluorescent staining as described under the Materials and Methods. Scale bar = 100 mm. D: MDA-MB231 cells were pretreated with SB203580 (5 mM) for 2 hours then placed into the upper compartments of migration chambers (transwells) in the presence of PIC (0 or 10 ng/ml). The number of MDA-MB231 cells that migrated through the transwells was determined as described under the Materials and Methods. The experiment was replicated three times. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated groups. doi:10.1371/journal.pone.0047721.g005 PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47721