Figure 3. Involvement of Rac pathway in HGF-induced MT-associated signaling.

A–D: HPAEC were subjected to pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) or knockdown of Rac1 or PAK1 as described in Methods and stimulated with HGF (50 ng/ml) for the indicated periods of time. A: Rac activation was determined by Rac-GTP pulldown assay. The content of activated Rac was normalized to the total Rac content in EC lysates. B: Time-dependent stimulation of stathmin phosphorylation and increase in tubulin acetylation was detected by western blot. C: Effect of preincubation with c-Met inhibitor on HGF-induced stathmin phosphorylation was evaluated by western blot with phospho-S⁶³-stathmin antibody. D and E: HGF-induced stathmin phosphorylation and tubulin acetylation in cells with Rac1 (D) and PAK1 (E) knockdown were evaluated by western blot. siRNA-induced target protein depletion was confirmed by membrane probing with Rac1 or PAK1 antibody. Equal protein loading in all assays was confirmed by membrane probing with β-actin antibody. F: Bar graphs depict the quantitative densitometry analysis of western blot data from four independent experiments; *P <0.05, RDU: relative density units.