Figure 5

Taxol dose-dependent increase in the association of Gαi1 protein with the microtubules exclusively in the 2008/13/4 cells. The 2008 and 2008/13/4 cells were seeded at a density of 1 × 10⁶/ml and incubated under normal growth conditions for 36 hours. Thereafter, the 2008 cells untreated (lane 1) or treated for 24 hr with 25 nM (lane 2) and 500 nM (lane 3) taxol and the 2008/13/4 cells untreated (lane 4) or treated for 24 hr with 500 nM (lane 5) and 5 μM (lane 6) and then washed with chilled PBS (3 ×). The cytoskeletal fraction was isolated essentially as described previously [22]. Briefly, the attached cells were incubated in a microtubule-stabilizing buffer (0.1 M PIPES, 1 mM EGTA, 1 mM MgSO₄, 2 M glycerol, pH 8.0) for 20 min. Thereafter, the cytosolic protein were removed by incubating the cells in the microtubule stabilizing buffer containing 0.1% NP-40 and protease inhibitor cocktail for 20 min. The cytoskeletal fraction (attached to the plastic dishes) was scraped into RIPA buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitor cocktail. The cytoskeletal fraction (5 μg protein/lane) was then subjected to SDS-PAGE. The separated proteins were transferred to PVDF membranes and Western blotting was performed using the polyclonal antibody against Gαi1.