FIG. 6.

Gab1 and SHP-2 overexpression increases NF-κB activity in glioblastoma cells via the PI3-kinase pathway. (A) U87MG cells and U87MG/T691 cells were cotransfected with a 5× NF-κB-luciferase reporter gene, the pSV-β-galactosidase vector, and one of the following constructs (0.5 μg each): an empty vector, a wild-type Gab1 construct, a wild-type SHP-2 construct, or a vector expressing a Gab1 mutant incapable of binding the p85 subunit of PI3-kinase (YF3). (B) LN229 cells were cotransfected with a 5× NF-κB-luciferase reporter gene, the pSV-β-galactosidase vector, and 0.5 μg of one of the following constructs: an active (myristoylated) Akt or a kinase-dead Akt construct, a wild-type Gab1 construct, a wild-type SHP-2 construct, a vector expressing a Gab1 mutant incapable of binding the p85 subunit of PI3-kinase (YF3), a vector expressing constitutive active EGFR (ΔEGFR), or a vector expressing the ErbB2/Neu receptor mutant (T691stop). In addition, U87MG, U87MG/T691, and LN229 cells were cotransfected with a 5× NF-κB-Luciferase reporter gene, the pSV-β-galactosidase vector, a wild-type Gab1 construct, and a vector expressing wild-type SHP-2 (A and B). Forty-eight hours after transfection, cells were serum starved for 24 h, followed by cell lysis and measurement of luciferase activity. Values obtained were normalized to β-galactosidase activity. The experiments were performed three times in duplicate. Error bars represents standard deviations. Basal promoter activity of the NF-κB-Luciferase reporter when transfected with empty vector alone is set at 1. P values were <0.05. (C) U87MG cells were transiently transfected with 5 μg each of the empty vector, Gab1(WT), Gab1(YF3), and SHP-2(WT) constructs. Twenty-four hours after transfection, cells were placed in serum-free medium. Forty-eight hours after transfection, cells were harvested and nuclear extracts were made. EMSAs were performed as described in Materials and Methods.