The kinetics of p38 and MAPK inhibitor ERK activation after induction were assessed by Western blotting using antibodies that specifically recognize the phosphorylated forms of p38 and ERK MAPKs. Active p38 was detected in PMA-differentiated U937 cells induced by PCN, but the activation was transient, appearing at 10 and 30 min and returned to baseline level after another 30 min. Exposure of PMA-differentiated U937 cells to PCN for 30 min reduced activation of ERK1/2. After 30 min of induction, activation

of ERK1/2 began to recover but then its activation was down-regulated in a time-dependent manner, while the total ERK, p38MAPK levels remained almost unchanged throughout the experimental period (Figure 7). Figure 7 The expression of phosphorylated and total MAPK proteins in PMA-differentiated

U937 cells. PMA-differentiated U937 cells were stimulated with PCN (50 μM) for the indicated time periods with or without pretreatment by MAPK inhibitor SB 203580 (30 μM) or PD98059 (30 μM ) for 1 h. (A and B) The expressions of phospho-ERK or ERK (A) and phospho-p38 or p38 (B). (C) The expression of phosphorylated and total p38 and ERK proteins in U937 cells. Representative data of three independent experiments are shown. **p < 0.01 check details compared with the A group; MAPK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase; PMA: phorbol 12-myristate 13-acetate. PCN stimulated U937 cells to activate NF-κB signaling pathway Activation of the NF-κB signaling pathway is frequently involved in the regulation of many immune response and inflammatory Thalidomide genes [27]. To determine whether PCN affects NF-κB signaling pathway, we examined the effect of PCN treatment on a series of molecular events that leads to NF-κB activation, including degradation of I-κBα protein, translocation of p65 to the nucleus, and the phosphorylation of p65. We used PCN (50 μM) to stimulate PMA-differentiated U937 cells. At 0, 10, 30, 60, 90, and 120 min, cell proteins were collected and NF-κB p65 protein translocation

was detected by Western blotting. As shown in Figure 8, within 10 min after addition of PCN, the level of p-I-κBα in the cytosol was increased, which returned to baseline level after 60 min. We further investigated the change in nuclear localization of p65 protein. Within 10 min after addition of PCN, the level of p-p65 in total cell lysate and cytosol was increased. There was also an increase in the levels of p-p65 in the nuclear extract, as evidenced by high levels of p-p65 which persisted in total cell lysates (Figure 8). These results suggest that PCN induces degradation of I-κBα and subsequent translocation of NF-κB to the nucleus. Figure 8 PCN activates NF-κB signaling pathway. Differentiated U937 cells were stimulated with PCN (50 μM). At 0, 10, 30, 60, 90 and 120 min, cell proteins were collected. Cytosolic or nuclear protein was extracted, and Western blotting was performed to detect NF-κB p65 protein translocation.