FIG. 2.

Validation of peroxiredoxin (Prdx) 1 and Prdx-4 expression in A549 cells by Western immunoblotting. (A) Effect of RSV infection on cytoplasmic-nuclear localization of Prdx-1 and Prdx-4 in A549 cells by Western blotting. Cells were infected with RSV for 0 to 24 h, and 50 μg of cytoplasmic (Cyt) or nuclear (Nuc) extracts were separated on a 12% SDS-PAGE gel and transferred to PVDF membranes. Membranes were probed with either Prdx-1 antibody (anti-Prdx-1 Ab) or Prdx-4 Ab (anti-Prdx-4 Ab) (left) or immunogen peptide-preadsorbed Prdx-1 and Prdx-4 Abs (right). After the membranes were stripped, the membranes were probed with either anti-Lam B, anti-β-tubulin, or anti-β-actin Abs. Molecular weight markers are shown on the left. (B) Induction of Prdx-1 by RSV infection. Nuclear extracts (150 μg) from uninfected (−RSV) or RSV-infected (+RSV) A549 cells were separated on a 2-D gel and transferred to PVDF membranes. Membranes were probed with Prdx-1 Ab (anti-Prdx-1 Ab). Arrows labeled “a” and “b” indicate dimeric and monomeric forms of Prdx-1, respectively. Molecular weight markers are shown on the left. (C) Prdx-1 Ab specificity. Identical amounts of proteins were separated by 2-DE and transferred to Immobilon membranes. Prdx-1 Ab was preadsorbed by mixing with blocking peptides before exposure to membranes. (D) RSV-induced changes of Prdx-4 by 2-DE Western blotting. Prdx-4 was detected by Western blotting using Prdx-4 Ab (anti-Prdx-4 Ab). The arrow labeled “a” designates dimer; the arrow labeled “b” designates the monomer of Prdx-4. (E) Prdx-4 Ab was preadsorbed prior to blotting, as described for panel C.