Fig. 4. Plant AP180ENTH binding to C-adaptin ear constructs. (A) Pull-down experiments with the full-length ear region or the truncated version of At-C-Ad and GST as a control together with the recombinant H₆-AtAP180ENTH construct show that binding requires the ear region. (B) Analysis of the consensus binding motifs of At-AP180 for binding to the C-ear. Binding experiments with alanine substitutions mutans of either the DPF motif (At-AP180ENTH-DPF/AAA) or the DPW-motif (At-AP180ENTH-DPW/AAA) with the full ear construct of At-C-adaptin reveal that the DPF-motif is crucial for binding. GST serves as a control. The antibody against histidine recognizes the AP180-constructs in immunoblots of (A) and (B). P, pellet; S, supernatant.