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. Author manuscript; available in PMC: 2017 Feb 21.
Published in final edited form as: Traffic. 2016 Dec 14;18(1):44–57. doi: 10.1111/tra.12457

Figure 6. The overlapping β2 adaptin arrestin-box and clathrin-box motifs both bind multiple sites on NTD.

Figure 6

(A) GST fusions of the CBM (GST-AP2CBM) and arrestin-binding motif (GST-AP2arrS and GST-AP2arrL) from the hinge region of β2 adaptin, the arrestin-box motif constructs having either the next residue of β2 adaptin (‘L’, GST-AP2arrS) or the sequence that follows the LLGDL motif of arrestin2L (‘ASS’, GST-AP2arrL) appended at their C termini. (B) Capture of wild-type NTD or a mutant with disrupted clathrin and Royle boxes (Q89A+F91K+F9W) by GST-tagged β2 adaptin clathrin-binding motifs. Purified recombinant wild-type or mutant His-NTD-NEMO was incubated with glutathione sepharose pre-loaded with GST-tagged “bait” proteins. After washing, proteins bound to the beads (pellet) were subjected to SDS-PAGE and immunoblotting (WB) using an antibody that recognizes clathrin NTD (αNTD).