Figure 6

PG27 and PG490 down regulated MAPK activity. T cells at a concentration of 5 × 10⁶/mL were pretreated with various concentrations of PG27 (A and B) or PG490 (C and D) for 2 h and then stimulated with PMA + ionomycin for 30 min (A), TNF-α for 10 min (B and C) or CD3/CD28 for 30 min (D). Cell pellets were collected and total cell lysates were immunoprecipitated with anti-JNK, anti-p38 or anti-ERK antibodies. After sequential washes, the substrates (GST-c-Jun for JNK and MBP for both p38 and ERK) and [γ-³²P]ATP were added individually. After the kinase reaction, the mixture was analyzed by SDS-PAGE. Representative data of at least 3 independent experiments are shown.