Fig. 5. SNX18 colocalizes and interacts with AP-1. (A) HeLa cells were stained for endogenous SNX18 and AP-1, and visualized by epifluorescence. Scale bar: 10 µm. The indicated boxed area was magnified and arrows highlight extensive colocalization in the periphery. (B) Pull-down experiment with GST and GST-fusion proteins containing ear domains of the mammalian AP complexes. Fusion proteins bound to beads were incubated with His-tagged LC-region from SNX18, and bound and unbound SNX18-LC was analyzed by SDS-PAGE and filter-blotted with His-probe (Pierce). The Coomassie-stained gel shows the fusion proteins. (C) Pull-down experiment with GST and GST-fusion proteins containing ear domains of 1 or the related subunit 2, or the GAE domains of GGA1 or GGA2. Pull-down was performed as in B. (D) Demonstration of a specific 1-binding site in SNX18. The alignment shows a conserved acidic patch in the LC region of SNX18. Black shade indicates conserved tryptophans and acidic residues, and gray shade indicates flanking amino acids with conserved properties in at least four out of five species. Bead-bound GST and GST-1-ear were incubated with wild-type (wt) or mutated (W154S and W158S, arrows in the alignment) His-SNX18-LC, and pull-down was performed as in B.