FIG. 3.

Interaction of BAX BH3 mutants in a transient transfection system. (A) NP-40-solubilized lysates from 293T cells transfected with Bax mutants and Bcl-2 were immunoprecipitated with 6C8 anti-BCL-2 or 12CA5 anti-HA MAbs. Immunoprecipitates (first panel, anti-BCL-2 IP; second panel, anti-HA IP) or direct cell lysates (third and fourth panels) were size fractionated onto 16% Tris-glycine SDS-PAGE gels, followed by the development of Western blots with the N20 anti-BAX Ab (Santa Cruz Biotechnology) or 6C8 anti-BCL-2 MAb. (B) NP-40-solubilized lysates from 293T cells cotransfected with Bax mutants that were either tagged with HA or lacking the COOH-terminal hydrophobic segment were immunoprecipitated with 12CA5 anti-HA MAb, followed by the same steps as those described for panel A. Upper panel, results of the IP-Western analysis; lower panel, results of Western analysis of total cell lysates.