RAW cells were incubated with or without 2 uM PD184352 for 1 hour and/or 100 nM wortmannin for 10 mins prior to being stimulated for 30 mins with 100 ng/ml LPS and then lysed. c-Jun was immunoprecipitated from the lysates using anti-c-Jun [S230B] and the immunoprecipitates were subjected to SDS-Page and immunoblotted with anti-c-Jun phospho Thr 239 [S678A], anti-c-Jun phospho Ser 243 and anti-c-Jun [S230B] Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).