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. 2013 Mar 5;8(3):e58335. doi: 10.1371/journal.pone.0058335

Figure 4. Extents of disulfide bond formation between Cys in S0 and Cys in S4.

Figure 4

. (A–C) Cells were transfected with the indicated double-Cys-mutant BK α. After 2 days, the cells were collected, and biotinylated with the impermeant sulfo-NHS-biotin. The cells were divided and were either not further treated, treated with 10 mM DTT, or treated with 10 mM DTT and 40 μM QPD. The conditions were the same as in Fig. 2. Cells were lysed. Solubilized BK α was captured on Neutravidin beads, cleaved with HRV-3c protease between S0 and S1, electrophoresed, and immuno-blotted with an anti-BK α-C-terminal-epitope antibody. The extents of crosslinking were calculated from the relative integrated densities of the full-length α band and the truncated (Frag) α band, corrected by the efficiency of HRV-3c cleavage, determined individually for each Cys pair in each experiment (not shown). The efficiencies of cleavage were approximately 70%. N = 2–4. Mean + SD. N = 2–4 experiments, each with duplicate determinations. * P<0.05, **P<0.01, *** P<0.001, ****, P< 0.0001 by one-way Anova followed by Tukey’s post-hoc analysis.