Phospho-ibuprofen-modulated mitogen-activated protein kinases in breast cancers. (A) Phospho-ibuprofen (P-I) dissociates apoptosis signal-regulating kinase 1 (ASK1)-thioredoxin-1 (Trx-1) complex in MCF-7 cells. Total cell lysates from vehicle-treated or P-I-treated cells were immunoprecipitated using anti-Trx-1 antibody in the presence (+) or absence (-) of dithiothreitol (DTT). Supernatants (IB) and immunoprecipitants (IP) were immunoblotted with anti-ASK1 and anti-Trx-1, respectively. IC50, concentration that inhibits cell growth by 50%. (B) Mitogen-activated protein kinase (MAPK) activity was determined by immunoblotting MCF-7 cell lysates with antibodies against phosphorylated or total MAPKs as indicated. (C) Effect of P-I on MAPK signaling in breast cancer xenografts. Protein lysates from MCF-7 xenografts (upper) or from MDA-MB231 xenografts (lower) as described in Figure 1 were immunoblotted with antibodies against phosphorylated MAPKs. Loading control, β-actin. Lipo-P-I, liposome-encapsulated phospho-ibuprofen. Results were quantified and graphed on the right. #P < 0.05; *P < 0.001.