Figure 5

Involvement of FAK and β₁ integrin subunit with the activation of Ras and ERK pathway in pancreatic cancer cells. Examination of Ras and downstream ERK activation was performed as described in Materials and Methods. Cell lysates were prepared according to the instructions provided in the Ras Activation Assay Kit, and affinity precipitation of GTP-bound Ras was performed using GST-tagged Raf-RBD. Levels of pull-downed Ras (Ras-GTP) were determined by anti-Ras immunoblotting.(A) AsPC-1, BxPC-3, and Capan-2 cells were serum starved for 24 h and then attached to Coll IV with 10 ng/ml IL-1α for 15, 30, or 60 min. The time-dependent Ras activation and ERK1/2 phosphorylation by adhesion to Coll IV was demonstrated. Detection of total ERK 1/2 levels served as a loading control.(B) AsPC-1, BxPC-3, and Capan-2 cells were serum starved for 24 h and then attached to Coll IV with 10 ng/ml IL-1α in the presence or absence of inhibitory antibodies against β₁ integrin subunit for 30 min. FAK siRNA transfected pancreatic cancer cells were attached to Coll IV with 10 ng/ml IL-1α. Effects of IL-1α, FAK gene silencing, and β₁ integrin blocking on the activation of Ras/ERK signaling pathway in pancreatic cancer cells were demonstrated. Detection of total ERK 1/2 levels served as a loading control.