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. 2011 Jan 1;286(10):8213–8230. doi: 10.1074/jbc.M110.165852

FIGURE 3.

FIGURE 3.

BAKΔC binds CL directly and preferentially over other anionic lipids. A, BAKΔC was incubated with freeze/thaw liposomes composed of 30BrPC/30PE/10PI/30CL (CL), 30BrPC/10PI/60PG (PG), 30BrPC/70PI (PI), 30BrCL/30 PE/10 PI/30 (LCL), or 30 BrPC/30PE/10PI/30mCL (mCL) followed by sedimentation of the liposomes by centrifugation and quantitative assessment of liposome-bound BAKΔC fraction by immunoblotting and densitometry. Each bar represents the average and S.E. of at least three independent experiments (except LCL, n = 2). B, BAKΔC immunoreactivity is shown from a representative protein:lipid dot-blot experiment in which BAKΔC was overlaid onto commercially available nitrocellulose membranes spotted with some of the most common glycerolipids and sphingolipids present in mammalian cells. TG, triacylglycerol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol triphosphate; SM, sphingomyelin; SULF, sulfatide. PIP2-grip represents an internal control protein that binds PIP2 with high affinity provided by the producer. C, quantitative data on BAKΔC binding potency for different anionic lipids was determined using protein-lipid overlays in which increasing amounts of indicated lipids were spotted on nitrocellulose. Integrated BAKΔC immunoreactivity was measured at each spot and normalized for each lipid, and the data were fitted with a sigmoidal dose-response nonlinear regression model. EC50 values for CL, PG, and PI were 411.3 ± 51.2, 2232.6 ± 87.0, and 5674.2 ± 123.1 pmol, respectively. D, representative sensorgrams depict the interaction of BAKΔC with LUVs containing the indicated lipids immobilized on the surface of L1 sensor chip. Liposome lipid compositions were as follows: MOM, 55PC/35PE/10PI; 7.5CL, 47.5PC/35PE/10PI/7.5CL; 15CL, 40PC/35PE/10PI/15CL; 30CL, 25PC/35PE/10PI/25CL; 60CL, 20PC/10PE/10PI/60CL; 7.5PG, 47.5PC/35PE/10PI/7.5PG; 15PG, 40PC/35PE/10PI/15PG; 30PG, 25PC/35PE/10PI/30PG; 60PG, 20PC/10PE/10PI/60PG. Inset, summary of association binding responses obtained in four independent SPR experiments corresponding to BAKΔC:15CL LUV and BAKΔC:30PG LUV samples. RU, resonance units.