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.
