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. Author manuscript; available in PMC: 2006 Nov 21.
Published in final edited form as: J Biol Chem. 2006 Mar 30;281(23):15941–15950. doi: 10.1074/jbc.M512586200

FIGURE 8. Overexpression of Bcl-xL inhibits vinblastine-induced Bax activation, Bax dimerization, and apoptosis.

FIGURE 8

A, transient expression of HA-Bcl-xL. KB-3 cells were untransfected, transfected with vector only (Vec), or with HA-Bcl-xL plasmid. Whole cell extracts were subjected to immunoprecipitation with anti-HA antibody (upper two panels) or anti-Bcl-xL antibody (bottom panel). Ab (+ or −) indicates whether antibody was present or omitted in the immunoprecipitation step. Immunoprecipitates were separated by SDS-PAGE and blotted with the antibody indicated on the right. B, stable expression of HA-Bcl-xL. KB-3 cells stably expressing HA-Bcl-xL were isolated, as described under “Experimental Procedures.” Extracts from untransfected (−) or two clones of HA-Bcl-xL transfected cells (+) were subjected to immunoblotting with antibody to either Bcl-xL or the HA tag as indicated. GAPDH provided a loading control. C, KB-3 (top panel) or KB3-HA-Bcl-xL (lower panel) cells were untreated or treated with 30 nM vinblastine (VBL) for the times indicated and subjected to immunoprecipitation with anti-Bax 6A7 antibody (lanes 3, 6, and 9), followed by immunoblotting for Bax. Precipitates prepared in the absence of 6A7 antibody (lanes 2, 5, and 8) and whole cell extracts (lanes 1, 4, and 7) were also examined as controls. The data shown for KB-3 cells are the same as that presented in Fig. 3A. D, KB-3 or two independent clones of KB3-HA-Bcl-xL cells were untreated or treated with vinblastine as indicated, permeabilized, and treated with digitonin in the presence of 1 mM DSP, and CHAPS-solubilized particulate fractions were subjected to SDS-PAGE in the absence of β-mercaptoethanol. Immunoblotting for Bax was performed, with the monomeric (21-kDa) and oligomeric (42-kDa) forms shown. E, KB-3 or two clones of KB3-HA-Bcl-xL cells were left untreated (−) or treated with (+) vinblastine (30 nM, 48 h), and the relative extent of apoptosis quantitatively was assessed, as described under “Experimental Procedures.” The results are shown as the means ± S.D. (n = 6) and are representative of two independent experiments.