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. Author manuscript; available in PMC: 2013 Jan 15.
Published in final edited form as: Free Radic Biol Med. 2011 Nov 9;52(2):410–419. doi: 10.1016/j.freeradbiomed.2011.10.495

Figure 5. Bcl-2 co-immunoprecipitates with V5-OGC from co-transfected CHO cells, GSH-MEE enhances the interaction.

Figure 5

A. CHO cells were co-transfected with Bcl-2 and V5-OGC (5μg each) lysed in Wahl buffer containing 0.1% TritonX-100 and 1 mM DTT, and immunoprecipitated (IP) with a monoclonal V5 antibody. Immune complexes were then resolved by SDS-PAGE, immunoblotted for V5 and Bcl-2. WCL= Whole cell lysate, IP =immunoprecipitate, −V5 antibody=Protein A/G agarose alone was used in the IP. B. Same as in (A), except at 24 h post-transfection cells were treated with 2 mM GSH-MEE for 4 h (and GSH-MEE was added to the IP buffer). Alternatively, 1 mM DTT was added to the IP buffer. C. Densitometry comparing pixel density of co-immunoprecipitated Bcl-2 with V5-OGC in the presence of either 1 mM DTT (Con) or 2 mM GSH-MEE. Data are normalized to the amount of V5 immunoprecipitated and are expressed as a percent of Bcl-2 immunoprecipitated in the presence of DTT (Con). Data was analyzed using a Student’s T-test, *p<0.05, n=9. D. Recombinant Bcl-2 and recombinant GST-OGC were co-immunoprecipitated with either anti-Bcl-2 (Bcl-2 IP) or anti-Slc25a11 (OGC IP) in 0.1% Triton X-100/Wahl buffer with 11.1 μM GSH. Immune complexes were resolved by SDS-PAGE, immunoblotted for Bcl-2 and Slc25a11 (OGC). E. Recombinant Bcl-2 and recombinant GST-OGC were co-immunoprecipiated with anti-Bcl-2 in either 0.1% Triton X-100/Wahl buffer with 10 μM GSH (−EA) or 0.1% Triton X-100/Wahl buffer with 10 μM GSH and 100 μM ethacrynic acid (+EA). Immune complexes were resolved by SDS-PAGE, immunblotted for Bcl-2 and Slc25a11 (OGC).