Figure 2

LTA-induced c-Jun phosphorylation is mediated through JNK in RBA-1. (A) Cells were pretreated with SP600125 (1 μM) for 1 h and then incubated with 50 μg/ml LTA for the indicated time intervals. Conditioned media were collected and assayed for proMMP-9 expression and activity by gelatin zymography. ProMMP-2 expression is shown as an internal control. (B) Cells were pretreated with SP600125 for 1 h and then incubated with 50 mg/ml LTA for 16 h. Total RNA was extracted and analyzed by RT-PCR. (C) Time dependence of LTA-stimulated JNK phosphorylation. RBA-1 cells were pretreated with SP600125 for 1 h and then treated with 50 mg/ml LTA for the indicated time intervals. Phosphorylation of JNK and c-Jun was determined by western blot using an anti-phospho-JNK or phospho-c-Jun antibody. (D) Cells were transfected with an empty vector (pcDNA3, as a control) or dominant negative mutant of JNK (ΔJNK) for 24 h, and then exposed to LTA (50 mg/ml) for 24 h. Cell lysates were assayed by western blot using an anti-GAPDH antibody as a control. Conditioned media were analyzed by zymographic analysis. Data are expressed as mean ± SEM (A, B, D) or mean (C) of three independent experiments (n = 3). ^#P < 0.01, as compared with the cells exposed to LTA alone. The figure represents one of three individual experiments.