Effect of IL-1α on the activation of Ras and ERK pathway in SW1990 cell lines. Activation of Ras and downstream ERK was performed as described in Materials and Methods. SW1990 cells were serum starved for 24 h and then treated with IL-1α in the presence or absence of inhibitory antibodies for 30 min. 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. Effect of IL-1α and α6, β1 integrin and uPAR inhibitory antibodies on the baseline/IL-1α-induced activation of ERK was also examined by immunoblotting. Detection of total ERK 1/2 levels served as a loading control.