Figure 4

Re-oligomerization of adiponectin in 2, 5, 10, and 15 mM total glutathione at a constant reduction potential of -120 mV. (A) Native PAGE of adiponectin oligomers after collapse and subsequent incubation in various concentrations of glutathione kept at a reduction potential of -120 mV. (B) Non-reducing denaturing SDS-PAGE analysis of disulfide-bonded dimers and reduced monomers of the same reactions. (C) Western blot analysis of adiponectin glutathionylation under non-reducing denaturing conditions. Anti-glutathione primary antibody was purchased from Arbor Assays. (D) Ponceau S stain of the blot in C prior to blocking. Purified bovine octadecamer was collapsed to trimers as described in Methods. After collapse and removal of DTT, adiponectin was allowed to re-oligomerize in the presence of 2, 5, 10, or 15 mM total glutathione concentrations held at a constant reduction potential of -120 mV. Experiments were performed in anaerobic conditions to minimize the oxidizing effects of atmospheric oxygen. At each time point, NEM was added prior to removal of samples from anaerobic chamber. α-GSH: anti-GSH antibody; GS-Monomer: glutathionylated monomer.