The effects of anti-TNFR1 antibody on the activation of co-cultured-U87 cells. Experimental details in co-culture were indicated in Figure 1. The anti-TNFR1 antibody (300 ng/ml) or anti-CD40 antibody (300 ng/ml) was pretreated in astrocytes 30 min and 1 h before co-culture. Expressions of TNFR1, activities of JAKs, STAT1 and CBP, and expressions of cytokine mRNA were determined in protein extracts and nuclear extracts by western blot and RT-PCR, respectively. (A) Expression of TNFR1 in co-cultured-U87 (left-upper panel) or -HMC-1 cells (left-lower panel), and expression of TNFR1 after inhibitor pretreatment (right panel). (B, C) Phosphorylations of Jak1/2 and STAT1 after inhibitor pretreatment. (D) Expression of CBP after inhibitor pretreatment. (E) Expressions of cytokine mRNA after inhibitor pretreatment. U87 cells, U87 cell culture alone; TNFR1, anti-TNFR1 antibody alone pretreatment in U87 cells; Co-culture, U87 cells co-cultured with HMC-1 cells; αCD40, anti-CD40 antibody pretreatment; αCD40 + TNFR1, anti-CD40 antibody and anti-TNFR1 antibody pretreatment before co-culture. *, Numbers below bands are values obtained from four independent experiments (n = 4) as the ratio of band density of each group versus those of total protein, actin or GAPDH using densitometry analysis.