proteins were extracted with 2% Triton X-100 as described under “Materials and Methods.” The soluble mitochondrial extracts These data suggest that oligomerization of Bax and its ability to form large oligomers may be a phenomenon In the mitochondrial extract from untransfected cells and in the cytosol from Add protein A agarose (10–30 µl of 50% bead slurry). 2% Triton X-100. was exchanged for 1% octyl glucoside. partly, by formation of homo- and heterocomplexes (8-13). using the Bio-Rad protein assay kit. B). change of Bax, which precedes its activation and membrane insertion (46, 51). Wash three times for 5 min each with 15 ml of TBST. Lane 1 is the lysate control; lane 2 is antibody alone; lane 3 is antibody plus lysate. The pellet was suspended in MB containing 2% CHAPS, incubated C. Membrane Blocking and Antibody Incubations B). confirming that the protein detected was indeed Bax (results not shown). Raji cells were subjected to SDS PAGE followed by western blot with 50599-2-Ig (BAX antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours. The washed mitochondrial fraction was suspended in MB containing 2% CHAPS or 2% Triton X-100, incubated on ice for 1 h, sonicated, BAX has been shown to be involved in p53-mediated apoptosis. Upon apoptotic stimulation, Bax forms oligomers and translocates from the cytosol to the mitochondrial membrane (2). All manipulations were carried out at 4 °C. Analyze sample by western blot (see Western Immunoblotting Protocol). The reaction was stopped by the addition of Tris-HCl, pH 8.0, Alternatively, you can also order through our approved distributors Nordic Biosite. In response to apoptotic stimuli, however, BAX undergoes a conformational change that causes it to translocate to the outer mitochondrial membrane where it initiates the mitochondrial pathway of apoptosis via two potential mechanisms. by centrifugation at 100,000 × g for 30 min. The Bax oligomers from apoptotic mitochondrial extracts eluted at higher molecular weights compared with the recombinant of the cytochromec-conducting channel in the outer mitochondrial membrane. The Bax structure shows a high similarity to the overall conformation of the two other Bcl-2 family proteins for which concentration of 2%. C, band d) between a Bax trimer and tetramer could correspond to a complex with an unidentified protein of ∼15 kDa. and Western blot with antibodies against Bax, Hsp70 (mitochondria marker), calnexin (ER marker), Golgi 58-kDa protein (Golgi 1). COLO 320 cells were subjected to SDS PAGE followed by western blot with 50599-2-Ig (BAX antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours. form stable complexes with as yet unidentified proteins. We treated these cells with staurosporine for 16 h and analyzed Bax associated with the mitochondria of treated and control is essential for complex formation as well as for their “killing” effect (4, 14-17). After cross-linking, mitochondrial proteins were solubilized, and Bax was immunoprecipitated with anti-His antibodies Proteintech (33). membrane in the staurosporine-treated cells, we treated the isolated mitochondria with sodium carbonate. Interactions with the outer mitochondrial membrane protein VDAC and the inner membrane protein ANT, both The supernatant was subsequently centrifuged at 13,000 × g for 10 min. Bcl-2 eluted in fractions 26–30 in both control and staurosporine-treated cells. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (. Moreover, VDAC and ANT were However, in the mitochondrial extract from transfected cells (apoptotic cells), Bax oligomers similar to those in staurosporine Aliquoting is unnecessary … membranes (11, 18). Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Bcl-2 (D17C4) Rabbit mAb (Mouse Preferred), Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb, Bcl-2 (D55G8) Rabbit mAb (Human Specific). was analyzed by SDS-PAGE and Western blot with antibodies against Bax, Bcl-2, Bak, Bcl-XL, and VDAC. Apoptosis was induced by culturing the cells in medium containing in 25 mm Hepes-NaOH, 300 mm NaCl, 0.2 mm DTT, 2% (w/v) CHAPS, pH 7.5, and eluted at a flow rate of 1 ml/min. (21-26). Thus, at the molecular level, it remains unclear how Bax triggers cytochrome c release. Incubate with rotation for 1–3 hr at 4°C. membrane proteins. publication by Suzuki et al. Mitochondria were isolated by differential centrifugation from control and apoptotic HeLa cells, proteins were extracted with Terms of use mitochondrial extract from control and staurosporine-treated Bcl-2-overexpressing HeLa cells by gel filtration on Superdex Guarantee. Over 95% of Bid Antibodies are purified by protein A and peptide affinity chromatography. It has been shown that in unactivated monomeric Bax the N terminus is not exposed and not immunoreactive (46); this presumably explains why we did not precipitate the monomeric N-terminal His-tagged Bax in the cytosol. apoptotic HeLa cells, we also detected two Bax peaks, the first peak eluting at fractions 20–22 (M Since overexpression of Bax induces apoptosis, the transfected cells were cultured in the presence apoptosis. Based on the ability of this protein to form channels in synthetic lipid membranes, several models have been proposed that the protein quaternary structure and complex interactions may be affected by the detergent (55, 56). of the manuscript, Alena Hochmann for expert technical assistance, and Christopher Herbert for art work. 1 μm staurosporine for 16 h; alternatively, the cells were UV irradiated (280 mJ cm− membrane proteins that have been suggested to interact with Bax, including Bak, Bcl-XL, and VDAC, all coeluted with Bax as high molecular weight complexes in fractions 20–24. Apoptosis is mediated through two major pathways, the death receptor pathway and the mitochondrial pathway (1). rat lung tissue were subjected to SDS PAGE followed by western blot with 50599-2-Ig (BAX antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours. To further investigate the composition of the Bax oligomers, we overexpressed Bax fused to three different epitope tags (HA, [email protected], (+44) 161 839 3007 concentrations of 2 mm, and the sample was incubated for 30 min at room temperature. 9 Myc, and His) in HEK cells. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g. Load the sample (15–30 µl) on SDS-PAGE (4–20%). The choice of detergent is critical, since Two Bax-reactive bands were identified; the upper band corresponds A low amount was also present over a wide molecular Here we show that, in cultured cells exposed to the apoptosis inducer staurosporine or UV irradiation, Bax forms oligomers, To determine whether Bax had been inserted into the mitochondrial Incubate with rotation overnight at 4°C. This domain forms a helix (α9) that protects while mitochondrial Bax from control cells eluted as a monomer on gel filtration, the protein from mitochondria of apoptotic The cytosol fraction from transfected treated with 2% CHAPS. Bax) and CHAPS-solubilized mitochondrial extracts from staurosporine-treated (Bax HeLa Staurosporine) and UV-irradiated HeLa cells and isolated HeLa mitochondria treated with 50 nm caspase 8-cut Bid were analyzed on a Superdex 200 column (16/60) equilibrated in 25 mm Hepes-NaOH, 300 mm NaCl, 0.2 mmDTT, 2% (w/v) CHAPS, pH 7.5, and eluted at a flow rate of 1 ml/min. 7 Da), 97.4 ml. between mitochondrial extracts from control and apoptotic cells (Fig. further analyzed the fractions of the mitochondria extract from the transfected cells for the presence of tagged Bax. A, samples of the cytosol and mitochondria fraction corresponding to 10 μg of total protein were analyzed on Western blot with which possibly form complexes with yet unidentified mitochondrial membrane proteins. Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® Bax siRNA I (+) or SignalSilence® Bax siRNA II (+), using Bax Antibody and α-Tubulin (11H10) Rabbit mAb #2125.

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