Figure 3. Limited trypsinolysis of arrestin mutants

Native arrestin (WT) and arrestin mutants were subjected to limited trypsinolysis as described in Materials and Methods. A: Representative tryptic reactions in the presence (+) or absence (-) of 3.8 μM 7PP were terminated and separated on 12% SDS-PAGE and stained with coomassie blue. Full-length arrestin (arrow) is most susceptible to cleavage at its C-terminus, resulting in a 44 kDa product (arrowhead); molecular mass markers indicated in kDa. B: Trypsinolysis rates were calculated for each of the mutants in the presence (dark bar) or absence (light bar) of 3.8 μM 7PP. Error bars represent SEM (n=3). C: Arrestins that demonstrated substantial sensitivity to 7PP were subjected to trypsinolysis for 20 min in the presence of a titration of 7PP (0-100 μM) to determine relative to the phospho-peptide.