FIG. 4.
Mitochondrion accumulation of Bim after Gadd45a induction increases physical association of Bim with Bcl-2 and enhances Bax multimerization. (A) After inducible expression of Gadd45a protein in the HeLa cell line, cells were collected at the indicated time points. Mitochondrial lysates were prepared (see Materials and Methods) to examine the interactions between Bim and Bcl-2 or Bcl-XL. Lysates were incubated with 10 μl of antibody to Bcl-2 or Bcl-XL and 20 μl of protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 4 h. Immunocomplexes were washed four times with lysis buffer and examined by immunoblot analysis for the existence of Bim and Bax. (B) After Gadd45a induction, cells were collected at the indicated time points and subjected to treatment with 1 mM DSP (cross-linking agent) at 37°C for 30 min before subcellular fractionation. Mitochondrial proteins were analyzed with antibodies to Bax.