Figure 5
Identification of IDRs and disulfide bridges in epitope NEP-D4.
(a) The disorder plot for epitope EP4 was predicted by GeneSilico metadisorder. The x-axis shows the residues from 18 to 133, and the y-axis shows the disordered tendency ranging from 0 to 1. All residues with a disorder probability of more than 0.5 were considered to be disordered. The residues with plotted lines in the light blue region were predicted to be disordered; their counterparts in the bluish white area were considered ordered. (b) Four GST-NEP-D4 samples were treated with Trypsin under the same conditions, and the reactions were stopped at 0 min, 1 min, 5 min and 15 min. Next, the samples were processed and analyzed by Western Blotting using anti-GST antibody and PN-D4 as the primary antibodies. (c) Similarly, four additional GST-NEP-D4 samples were treated with freeze (−80 °C) and thaw (room temperature) cycles, which were repeated up to 0, 1, 5 and 10, times, respectively. Next, these samples were analyzed by Western blotting using the same antibodies above. (d) To rule out the interference of disulfide bridges, the reduced (1 M DTT-treated) and non-reduced (non DTT-treated) GST-NEP-D4 were analyzed by Western blotting using PN-D4.
