Interaction of cargo proteins with the AP-1 core in the absence or presence of Arf1∆1-16-Q71L was analyzed using a previously described pull-down assay (Guo et al., 2013 (link); Ren et al., 2013 (link)). In brief, MBP-tagged cargo protein (20 μg) immobilized on amylose resin (New England Biolabs, Beverly, MA) was incubated with 0.05 μM AP-1 core precleared on amylose resin in the absence or presence of 10 μM Arf1∆1-16-Q71L and 1 mM GTP. The pull down was analyzed by SDS–PAGE, followed by immunoblotting.
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Cargo-AP-1 Interaction Assay
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Other organizations : Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health
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Variable analysis
independent variables
- Presence or absence of Arf1∆1-16-Q71L
- Interaction of cargo proteins with the AP-1 core
- Concentration of MBP-tagged cargo protein (20 μg)
- Concentration of AP-1 core (0.05 μM)
- Concentration of Arf1∆1-16-Q71L (10 μM)
- Presence of 1 mM GTP
- Positive control: Interaction of cargo proteins with the AP-1 core in the presence of Arf1∆1-16-Q71L and GTP
- Negative control: Interaction of cargo proteins with the AP-1 core in the absence of Arf1∆1-16-Q71L and GTP
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