Figure 3

Effects of inhibitors on activation of Jak 1/2 or STAT1 in co-cultured-U87 cells. Experimental details in co-culture were indicated in Figure 1. The anti-CD40 antibody (300 ng/ml), Jak inhibitor (10 μM AG490), 8-oxo-dG (300 μg/ml), PKC inhibitors (5 nM Gö6976 or staurosporine) or inhibitors of MAP kinase (50 μM PD98059 for ERK, 10 μM SP600125 for JNK, 10 μM SB203580 for p38) was pretreated in astrocytes 1 h, 5, 10 or 10 min, respectively, before co-culture, and CD40 siRNA was transfected, as described in "Methods". Activities of JAKs and STAT1 were determined in protein extracts by western blot using densitometry analysis. (A, B) Time courses for activities of Jak 1/2 and STAT1 after co-culture. (C, D) Activities of Jak 1/2 and STAT1 after inhibitors pretreatment. (E, F) Phosphorylation of STAT1 after pretreatment with PKC and MAP kinase inhibitors. *, Numbers below bands are values obtained from four independent experiments (n = 4) as the ratio of band density of each group versus that of total protein using densitometry analysis.