Fig. 3. Time-course of LPA-induced Ras, RhoA and Rac activation. PANC-1 cells were incubated overnight in DMEM without supplements and then treated for the indicated time periods with 10 µM LPA. GTP-bound GTPases were recovered from RIPA-cell lysates (0.5 mg protein for Ras detection, 1 mg protein for Rac detection, or 2 mg protein for K-Ras, N-Ras and RhoA detection) using GTPase-specific GST-fusion proteins in in vitro precipitation experiments. Proteins were separated by 12.5% SDS-PAGE and the amounts of bound GTP-Ras, GTP-Rac and GTP-RhoA proteins were analyzed in immunoblots (left panel). Pan-Ras antibody was used to detect all Ras-isoforms, and antibodies specifically reactive against N-Ras, K-Ras, Rac and RhoA were used to detect the individual GTPases. The right panel shows aliquots (30 µg of protein for Ras, 50 µg of protein for Rac, and 80 µg of protein for RhoA) of total cell lysates to control for equal GTPase loading. For direct comparison, activation of N- and K-Ras was determined in the same lysate. One blot representative for at least three independent experiments for each GTPase is shown.

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