Figure 3. Binding of Arf1 and Arf1... | Rockefeller University Press

$Figure 3. Binding of Arf1 and Arf1 variants to synthetic liposomes. (A) Comparison of the binding ability of Arf1-wt, Cys-wt, and Cys-Y35A to synthetic liposomes in the absence and presence of GTP. Arf1 variants were mixed with liposomes to a final concentration of 1.5 µM protein and 0.5 mM lipid in the presence of ARNO in a total volume of 100 µl. After 15 min of incubation at 37°C with or without 1 mM GTP, the samples were floated to an interface between 25 and 0% (wt/vol) sucrose. 10% of the collected liposomal fractions was compared with 5% of the input and analyzed for the presence of Arf1 and the Arf1 variants by SDS-PAGE and Western blotting, respectively. non-myrArf1, nonmyristoylated Arf1; myrArf1, myristoylated Arf1. (B) Cross-linking of the Cys variants using BMH. Purified Arf1 protein was mixed with BMH in a molar ratio of 2:1 and incubated for 1 h at RT. Thereafter, the cross-linking reaction was quenched by the addition of DTT and analyzed by SDS-PAGE and Coomassie staining. (C) Analysis of the binding ability of cross-linked Cys-wt (cl-Cys-wt) and Cys-Y35A (cl-Cys-Y35A) to synthetic liposomes in the absence and presence of GTP. The assay was performed as outlined in A. I, input; L, liposome-bound fraction.$

Figure 3.

Binding of Arf1 and Arf1 variants to synthetic liposomes. (A) Comparison of the binding ability of Arf1-wt, Cys-wt, and Cys-Y35A to synthetic liposomes in the absence and presence of GTP. Arf1 variants were mixed with liposomes to a final concentration of 1.5 µM protein and 0.5 mM lipid in the presence of ARNO in a total volume of 100 µl. After 15 min of incubation at 37°C with or without 1 mM GTP, the samples were floated to an interface between 25 and 0% (wt/vol) sucrose. 10% of the collected liposomal fractions was compared with 5% of the input and analyzed for the presence of Arf1 and the Arf1 variants by SDS-PAGE and Western blotting, respectively. non-myrArf1, nonmyristoylated Arf1; myrArf1, myristoylated Arf1. (B) Cross-linking of the Cys variants using BMH. Purified Arf1 protein was mixed with BMH in a molar ratio of 2:1 and incubated for 1 h at RT. Thereafter, the cross-linking reaction was quenched by the addition of DTT and analyzed by SDS-PAGE and Coomassie staining. (C) Analysis of the binding ability of cross-linked Cys-wt (cl-Cys-wt) and Cys-Y35A (cl-Cys-Y35A) to synthetic liposomes in the absence and presence of GTP. The assay was performed as outlined in A. I, input; L, liposome-bound fraction.

This Feature Is Available To Subscribers Only