Figure 5
Mutations that change hydrophobicity of the C-terminal region of ricin A-chain influence its interaction with EDEM1 and EDEM2. (A) His-tag pull-down assay with RTA-His, RTADHF-His and RTAIHF-His (0.5 μg each) and lysates from HEK293 cells overexpressing EDEM1-HA, EDEM2-HA or cells transfected with an control vector, Ctrl. Proteins bound to the Ni-NTA beads were analyzed by SDS/PAGE and Western blot with anti-HA antibodies (upper panel). The same membranes were re-probed with anti-RTA antibodies (middle panel). Whole cell lysates (WCL) analyzed with anti-HA antibodies are shown in the bottom panel. A representative experiment is shown. (B) Average data from His-tag pull-down with s.d. from four independent experiments. The amount of EDEM1 bound to RTADHF or RTAIHF is relative to the amount of EDEM1 interacting with wild-type RTA which is marked as 1. Data were analyzed by Student’s t-test , *P < 0.001, • P = 0.112 (C) Average data from His-tag pull-down with s.d. from four independent experiments. The amount of EDEM2 bound to RTADHF or RTAIHF is relative to the amount of EDEM2 interacting with wild-type RTA which is marked as 1. Data were analyzed by Student’s t-test, *P < 0.001, • P = 0.155.