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. 2017 Jun 7;7:2932. doi: 10.1038/s41598-017-03279-7

Figure 3.

Figure 3

Homo- and hetero-oligomerization of BB0323 and BB0238 in the presence and absence of BB0238 11-residue interaction region. (A) Quaternary structure of BB0238 and BB0238∆IM as assessed by analytical size exclusion chromatography. Elution profiles of various concentrations of proteins, 9 μM (dashed line), 18 μM (dotted line) and 44 μM (solid line) BB0238 (left panel) and 10 μM (dashed line), 20 μM (dotted line) and 40 μM (solid line) BB0238∆IM (right panel) from Sephadex 75 column. Lower panels show coomassie-stained denaturing SDS-PAGE of the eluted fractions F9-F14 from the experiments using 44 μM BB0238 or 40 μM BB0238∆IM. (B) BB0238 Cross-linking studies. Coomassie-stained denaturing SDS-PAGE of ~9 μM BB0238 and BB0238∆IM cross-linked with DMS as described in Materials and Methods. Lanes labeled (−) and (+) correspond to proteins that were treated with 1 M Triethanolamine buffer alone or with DMS, respectively. The right lane shows molecular weight markers. (C) Quaternary structure of BB0323 (residues 22–242) as assessed by analytical size exclusion chromatography. Elution profiles of 8 μM BB0323 from Superdex 75 column. Bottom panel showed coomassie blue-stained denaturing SDS-PAGE of BB0323 N2 in 0.5 mL fractions collected. Inset: Coomassie blue-stained denaturing SDS-PAGE of ~19 μM BB0323 crosslinked with DMS as described in Materials and Methods. BB0323 in the negative control (−) and positive (+) lanes were treated with 1 M Triethanolamine buffer alone or with DMS, respectively. (D) One dimensional PAGE separation of native BB0238 and BB0238∆IM. Gel separations were stained with Comassie Brilliant Blue (left panel), immunoblotted with anti-BB0238 antibody (middle panel) and subjected to far-Western analysis using anti-BB0323 antibody (right panel). (E) Elution profiles of 10 μM BB0238- BB0323 complex at ~1:1 molar ratio (solid line), 30 μM BB0323 (dotted line) and 40 μM BB0238 (dashed line) from Superdex 200 column with 1 mL fractions collected. (F) Elution profiles of ~9 μM BB0238∆IM-8 μM BB032322-242complex (solid line), 8 μM BB0323 (dotted line) and 9 μM BB0238∆IM (dashed line) from Superdex 75 column with 0.5 mL fractions collected.