Roflumilast n oxide

Roflumilast N-oxide (RNO) was provided by Nycomed (Konstanz, Germany). F-12K Nutrient Mixture Kaighn's Modification cell culture medium, antibiotics, glutamine and trypsin-EDTA were purchased from Invitrogen (Eugene, OR, USA). Fetal calf serum (FCS) was from Hyclone (Logan, UT, USA). Lipolysaccharide from E. coli 055:B5, Thiazolyl Blue Tetrazolium Blue (MTT), SB-203580, SP-600125 and U0126 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific antibodies against phospho-(p44/42) ERK1/2, (p44/42) ERK1/2, phospho-p38 MAP kinase, p38 MAP kinase, phospho-SAPK/JNK, SAPK/JNK, were purchased from Cell Signaling Technology (Beverly, MA, USA). Acrylamide, SDS, Tris, HEPES and BSA were purchased from Eurobio (Les Ulis, France). Bradford protein assay and precision plus protein dual color standards were purchased from Bio-Rad (Hercules, CA, USA).

Lipopolysaccharide (from E. coli serotype 0111:B4), formoterol fumarate dehydrate, DMSO, acetylcholine and histamine were purchased from Sigma (St. Louis, MO, USA). Roswell Park Memorial Institute 1640 medium, penicillin-streptomycin stabilized solution, and L-glutamine were purchased from Eurobio Biotechnology (Les Ulis, France). Roflumilast and roflumilast-N-oxide were synthesized by Nycomed GmbH (Konstanz, Germany). All other chemicals were of analytical grade and were obtained from Prolabo (Fontenay sous Bois, France). All cell culture plastics were from CCL (Nemours, France). Stock solutions of the PDE4 inhibitors were prepared in DMSO at 10 mM. A 2.5 mM formoterol stock solution was prepared in 10% DMSO. All subsequent dilutions were prepared daily in complete culture medium.