FIGURE 1.
NPC1L1 C terminus binds Numb, but not ARH or Dab2. A, schematic illustration of the in vitro pulldown assay. Recombinant His6-tagged CLASPs (ARH, Dab2, and Numb) or/and their PTBs were bound to Ni-NTA beads and tested for interaction with GST fusion proteins of either the NPC1L1 or LRP C terminus (positive control). B, purified recombinant proteins from E. coli for the binding assay shown by SDS-PAGE and Coomassie Brilliant Blue staining. C, interactions between His6-tagged CLASPs and GST-fused NPC1L1-C or LRP-C analyzed by in vitro pulldown. The specifically bound proteins (pellet) were assessed by Western blots with anti-GST antibody compared with input proteins (Input). D, increasing concentrations (50, 100, and 150 μm) of CLASPs were used in a pulldown assay. E, binding of Numb to GST-fused NPC1L1(H1309P)-C and mNPC1L1-C. IB, immunoblot.