Fig. 6. Hrs and Hbp interact with ubiquilin in a UIM-dependent manner, whereas epsin does not interact with ubiquilin. (A) Equal amounts of GST or GST-ubiquilin immobilized on glutathione-Sepharose beads were incubated with total cell lysates of Cos-1 cells transfected with GFP-Hrs or His₆-epsin (5% of the initial input is shown). The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-ubiquitin anti-GFP antibody (-GFP). The membrane was stripped and reprobed with anti-His₆ antibody (-His₆). (bottom) Coomassie staining of the membrane. The positions of GST and GST-ubiquilin are indicated. (B) GST-ubiquilin immobilized on glutathione-Sepharose beads was incubated with total cell lysates of Cos-1 cells transfected with GFP-Hrs wild type (WT), GFP-Hrs-UIM or GFP-Hrs-LSAA in which the two essential UIM residues L269 and S270 are mutated to alanine. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-GFP antibody (top, GST pull down). (bottom) Anti-GFP immunoblot of total cell lysates (5% of the initial input) showing similar expression levels of the GFP-Hrs constructs. (C) GST-ubiquilin immobilized on glutathione-Sepharose beads was incubated with total cell lysates of Cos-1 cells transfected with HA-Hbp wild type (WT) or HA-Hbp-LSAA in which the two essential UIM residues L176 and S177 are mutated to alanine. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-HA (top, GST pull down). (bottom) Anti-HA immunoblot of total cell lysates (5% of the initial input) showing similar expression levels of the HA-Hbp constructs. GST alone did not bind the HA-Hbp constructs (not shown). (D) Equal amounts of GST, GST-Hrs-CT (amino acids 1-454), GST-Hrs-CT-UIM or GST-Eps15-UIM 1+2 (amino acids 847-897) was incubated with total cell lysates of Cos-1 cells transfected with GFP-UBL. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-GFP antibody. The pulled-down GFP-UBL is indicated with an arrow. The asterisk (^*) indicates a degradation product of the UBL. (bottom) Coomassie staining of the membrane. The GST-Hrs-CT and GST-Hrs-CT-UIM (at the top of the membrane) displayed several degradation or incompletely translated products.