Ras Activity Assay

Ras activity was measured using Ras Activation Assay Kit (Millipore, USA) according to the manufacturer’s protocol. Briefly, cells were homogenized in 1× lysis buffer on ice, then sonicated and centrifuged at 12,000 rpm at 4°C for 10 min. Protein concentration was determined by the BCA Protein Assay Kit. Then 1/10 of the lysate was set aside to allow quantification of total Ras and protein concentration. GAPDH was used as an internal control. Equal amounts of lysate were incubated for 1 h at 4°C with agarose beads coated with the Raf-Ras binding domain provided in the kit. Beads were washed three times with ice-cold lysis buffer, and then boiled for 5 min with loading buffer.