Fig. 6. Raf-specific mutant forms of Ras promote NBT-II cell migration. Two days after seeding, cells were microinjected with 100 ng/µl of the Ras expression vectors and 25 ng/µl of the EGFP construct. Four hours after microinjection, cells were placed on the motorised stage of a Leica inverted microscope and recorded by videomicroscopy. Velocities were obtained by tracking individual EGFP-positive cells. Each column represents the mean of at least three independent experiments in which at least 40 cells were recorded. (A,B) Clusters of NBT-II cells were transiently transfected with the indicated constructs along with the EGFP reporter construct prior to videocinematography recording. Velocities were measured by tracking at least 40 EGFP-positive cells. For each construct, at least three independent experiments were performed. The mean velocity and s.e.m. are shown. (C) The Rac pull-down assay was done on NBT-II cells transiently transfected with the indicated constructs. The upper panel shows a Rac immunoblot performed on cell extracts purified on PAK-GST Sepharose beads. The lower panel shows a Rac immunoblot of total cell extracts.