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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Cytoskeleton (Hoboken). 2014 Feb 26;71(4):257–272. doi: 10.1002/cm.21167

FIGURE 6.

FIGURE 6

Rac1 undergoes activation in cells expressing S165D-α6-tubulin. (A) Evidence of activated Rac1 in membrane ruffles of MCF-10A cells. Immunofluorescence of cells treated either with DMSO (0.05% v/v, control) or DAG-lactone (10 μM, 1h) was performed followed by fixation with 4% PFA and addition of anti-active Rac1 antibody (1:100). With DAG-lactone treatment, Rac1 localized to membrane ruffles (arrows). Scale bar, 10 μm. (B) Western blot analysis of the level of GTP-bound Rac1, Cdc42, or RhoA in response to S165D or S165N α6-tubulin mutants, or the vector control (VC). The immunoblot shows the results of pull-down assays with whole cell lysates (600 μg per sample), as described in ‘Methods’. The results are representative of three independent experiments. (C) Motility of MCF-10A transfectants expressing wildtype PKCα, S165D-α6-tubulin, or the empty vector (VC) was measured with or without the Rac1 inhibitor NSC23766 (75 μM) (in water). Measurements show the area occupied by the cells in triplicate samples after 8 h of treatment (‘Methods’). (*, p<0.0001) The results are representative of three independent experiments.