Fig. 5. Rac1 activation by downstream pathways of Ras. (A) NBT-II cells were
activated with 30 ng/ml of EGF for the indicated times, then lysed with lysis
buffer as indicated in the Materials and Methods. Cell extracts were incubated
with GST-PAK2 Sepharose beads, washed and immunoblotted with anti-Rac1
antibodies. Levels of endogenous Rac were estimated by immunoblotting total
cell extracts with Rac1 antibodies. Alternatively, equivalent amounts of cell
extracts were immunoprecipitated with anti-MAPK antibodies and subjected to a
kinase assay using MBP as an exogenous substrate. (B-D) Rac pull-down was done
on NBT-II cells transiently transfected with the indicated constructs. The
upper panel shows the amounts of Rac bound to PAK-GST beads as revealed by
Rac1 antibodies. The middle panel shows the levels of Rac as estimated by Rac
immunoblotting on total cell extracts. The lower panel shows the levels of
EGFP expression measured by EGFP immunoblotting on total cell extracts with
anti-GFP antibodies.
