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. 2018 Apr 23;293(23):8982–8993. doi: 10.1074/jbc.RA118.002766

Figure 2.

Figure 2.

Assessment of the stability of membrane-associated PopB and PopD. A, PopB and PopD remained bound to membranes after pH neutralization. Purified PopD and PopB (1:7 molar ratio) were added to liposomes suspended in 20 mm sodium acetate, pH 4, or buffer C (HBS). Sample neutralization was achieved by addition of 2 m Trizma base. Membrane-associated proteins were separated from unbound proteins using a membrane flotation assay. The top fractions containing membrane-bound proteins were collected, precipitated with TCA, and analyzed for PopB and PopD by immunoblotting. Band intensities were quantified using ImageJ. Membrane-bound proteins in lanes labeled 7.5, 4–7, and 4 (no liposome) were quantified by normalizing the detected PopB and PopD to that in lane 4 (plus liposomes). Data are reported as the mean from two independent experiments and their range. Representative blots are shown. B, some dissociation of PopD from homo-oligomers was observed when proteoliposomes were subjected to the indicated treatments. Proteoliposomes containing PopD alone were treated with buffer C, 6 m urea, pH 8.0, 0.1 m sodium carbonate, pH 11.5, or 1 m NaCl in 10 mm Hepes, pH 7.5, for 30 min on ice. Membrane-bound proteins (top fractions: T) were separated from dissociated proteins (middle: M, and bottom: B fractions) using a floatation assay. All fractions (T, M, and B) were collected, precipitated with TCA, and PopD was detected by immunoblotting. The amount of dissociated proteins was quantified and shown as the percentage of total protein ((M + B)/(M + B + T)) in each condition. Data are reported as the mean from two independent experiments and the range. Representative blots are shown. C, PopB and PopD hetero-complexes were stably inserted in the membranes. Extraction of PopB and PopD on liposomes were performed as described in B. PopB and PopD were detected by immunoblotting using the indicated primary antibodies and peroxidase-conjugated secondary antibodies. The amount of dissociated protein was analyzed as described in B.