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. 2015 May 31;290(30):18817–18832. doi: 10.1074/jbc.M114.612366

FIGURE 1.

FIGURE 1.

Gel filtration chromatography analyses of Rab complexes with FIPs. A, superimposed elution profiles of MBP-FIP2(410–512) (dashed line), Rab14(2–175) (dotted line), and the complex (solid line) are shown from a Superdex 200 16/60 column. B, elution profiles of wtRCP(581–649) (dashed line), Rab14(2–175) (dotted line), and the complex (solid line) are shown. C, preformed Rab14(2–175): wtRCP(581–649) complexes (Input) were incubated with Rab25(7–180) (Input) and re-loaded onto a gel filtration column (Superdex 200 16/60, GE Healthcare). The resulting two peaks (Output lanes) reveal displacement of Rab14 and the stable Rab25(7–180), wtRCP(581–649) complex. D, excess amounts of Rab11(1–173) and Rab14(2–175( (160 μm each) were incubated with a limiting amount of wtRCP(581–649) (60 μm) in a total volume of 500 μl. The sample was subjected to gel filtration chromatography (Superdex 200 10/300GL) and analyzed by SDS-PAGE (18% gel, visualized by Coomassie Blue). Solid lines are three fractions covering the complex, and dotted lines mark the fractions covering the excess (uncomplexed) Rabs. Loading controls are shown as follows: lane 1 is purified Rab11(1–173); lane 2 is Rab14(2–175); and lane 3 is wtRCP(581–649) (7 μl of a 60 μm solution of protein were loaded in each lane).