(A) There was an increased amount of
Raf-1 protein associated with the membrane in R2 over expressing cells.
The recombinant R2-expressing cell lines B3/mR2, N3/mR2, C1/mR2,
r-2/mR2, r-3/mR2, and NR4/mR2 (R2) were compared with their respective
control lines, B3/SH, N3/SH, C1/SH, r-2/SH, r-3, and NR4 (control). In
all cases, cells expressing recombinant R2 exhibited increased
membrane-associated Raf-1 protein, and when the two groups of cell
lines were compared, they were found to be significantly different by
t test analysis (P < 0.001).
(B) There was also an increase in the activity of MAPK-2 in
R2 overexpressing cells. The recombinant R2-expressing lines B3/mR2,
N3/mR2, 10T/mR2, C1/mR2, r-2/mR2, and NR4/mR2 (R2) were compared with
their respective control lines infected with LXSH (controls). In all
cases tested, cells expressing recombinant R2 showed increased enzyme
activity, and the difference between two groups was highly significant
(P < 0.001). (C) Increased focus
formation with N3/mR2 cells compared with N3/SH cells after
transfection with the activated V12 Rac-1 plasmid. The difference was
highly significant (P < 0.001). The number of foci
shown represents the average ± SE from two independent
experiments.