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. 2017 Aug 1;292(37):15287–15300. doi: 10.1074/jbc.M117.805044

Figure 3.

Figure 3.

The DTM can accommodate more PEX5(1–197;C11A) molecules than full-length PEX5(C11A). A, increasing volumes of RRLs (volumes in μl indicated at the top of each lane) containing either PEX5(1–324;C11A) or PEX5(C11A) were used in PNS-based in vitro reactions (“rx”) performed in the presence of ATP for 45 min. Organelle suspensions were then treated with PK and analyzed by SDS-PAGE/phosphorimaging. Total organelle samples (PEX5(1–324;C11A)) or one-third of them (PEX5(C11A)) were loaded onto the gel. The phosphorimage (upper panel) and a section of the corresponding Coomassie Blue-stained gel (middle panel) are shown. Numbers to the left indicate the molecular mass (kDa) of protein standards. Lower panel, data from the phosphorimaging quantitation were normalized for protein loads and methionine number in each radiolabeled protein (see supplemental Fig. S1 for details), fitted to a dose-response one-site-specific binding curve, and divided by the Bmax value obtained for full-length PEX5(C11A). Thus, values in the ordinate represent molar ratios of PEX5(1–324;C11A) to PEX5(C11A) (averages and standard deviations (error bars) from four replicates are shown). Bmax values for PEX5(1–324;C11A) and PEX5(C11A) are 1.21 (95% confidence interval (CI) = 0.88–1.99) and 1.00 (95% CI = 0.85–1.18), respectively. Note that the abscissa scale is different for the two radiolabeled proteins. B, exactly as in A but using radiolabeled PEX5(1–197;C11A) and PEX5(C11A). Bmax values for PEX5(1–197;C11A) and PEX5(C11A) are 2.52 (95% CI = 1.82–4.46) and 1.00 (95% CI = 0.90–1.11), respectively. In A and B, lanes In, RRL containing the indicated 35S-labeled proteins. Brackets and arrowheads indicate the PK-cleaved and intact PK-resistant 35S-labeled proteins, respectively. Note that intact PEX5(1–324;C11A) and intact PEX5(1–197;C11A) data cannot be fitted to the same dose-response curve (“ambiguous fit”); in these cases, the lines simply connect averages. Note also that for some points the error bars are shorter than the height of the symbols.