Figure 4.

Ent1 and Ub-Ent1 PIPs binding assays in vitro. A 1:1 mix of purified apo and ubiquitylated Ent1_1-184 proteins was assayed for PIPs binding. A PIP array membrane (Echelon) was incubated with the protein mix and washed, as described in the Methods section. Proteins were detected by western blot (WB): apo with anti-His₆-tag, and ubiquitylated Ent1 with anti-HA antibodies, respectively. (a) The PIP-array membrane after a binding assay with the mix and WB against the ubiquitylated Ent1. (b) The antibody reaction against ubiquitylated Ent1 that was blotted on the membrane, as described in the Results section. (c) The reaction with the HA antibody. The binding to the indicated PIPs is clearly seen as black spots. (d) A PI(4,5)P₂ enriched liposome (Echelon) binding assay. A protein mix contained apo and ubiquitylated Ent1_1-184 was used for a pull-down assay with PI(4,5)P₂ enriched liposomes. Following incubation, the liposomes were pelleted by centrifuge. Bound (Pellet) and unbound (Supernatant) proteins were then separated by SDS-PAGE, and detected by WB with anti-HA antibodies.