Figure 10.

Impairment of the interaction between the RLD1 domain and the clathrin heavy chain (CLThc) by the tambaleante (tbl) mutation. GFP fusion proteins with the amino acid residues of HERC1 RLD1 domain are shown. The pFG44 construct (RLD1 tbl) has a point mutation, resulting in a Gly → Glu substitution at 483 amino acid residue of HERC1. Pull-down experiments were performed in HEK-293 T cells transfected with pFG41, pFG44 or GFP fusion constructs. After 72 h post-transfection, cell lysates were incubated with GFP-Trap Agarose resin for 2 h. Proteins retained in the resin were analyzed by immunoblotting using specific antibodies against the indicated proteins (see Supplementary information). Note the absence of interaction between HERC1 and pFG44 construct.