FIGURE 3.
TolQ dimerizes through TMH2-TMH2 contacts. A, 0.4 × 108 Tuner (DE3) pET-Q cells producing the indicated TolQ8H variant were incubated (lower panel) or not (upper panel) with 0.3 mm CuoP before treatment with the thiol-blocking agent NEM. Heat-denatured samples were loaded on a non-reducing 12% acrylamide SDS-PAGE gel and analyzed by Western blot immunodetections using anti-His5 mAb. Prestained molecular mass markers (in kDa) and TolQ monomers (Q) and dimers (Q2) are indicated. B, about 3 μg of n-dodecyl-β-d-maltoside purified TolQ8H157C treated (+) or not (−) with the reducing agent β-mercaptoethanol (red) was loaded on a 12.5% acrylamide SDS-PAGE gel and analyzed by Coomassie Blue staining.