FIG. 3.

Inducible expression of Gadd45a causes dissociation of Bim from cytoskeleton. HeLa Gadd45a-inducible cells were cultured in DMEM containing tetracycline at a concentration of 2 μg/ml. At 24 h after withdrawal of the tetracycline, cells were treated with 5 μM Taxol (Sigma) for 4 h and 0.5 mM AMP-PNP for 30 min before lysis. Cellular protein extracts were prepared and fractionated by sucrose gradient sedimentation (see Materials and Methods). Proteins in gradient fractions were size separated by gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with antibodies to Gadd45a, Bim, LC8, and β-tubulin.