Fig 4. IMC3 is a direct binding partner of ILP1.

(A) ILP1-3xHA amber mutants yielding the two large upshifted bands (T184, T187, I188, and E209) were expressed in an endogenously tagged IMC3-3xMyc background that also contains the synthetase/tRNA pair. Following Azi addition and UV irradiation, the uncrosslinked (blue arrow) and crosslinked species (black asterisks) can be reproduced in this strain. When probing for IMC3-3xMyc, uncrosslinked protein is observed (blue arrow), but a persistent high molecular weight background prevents confirmation of any potential upshift of IMC3. (B) Strategy for denaturing IP. Boiling in SDS disrupts the parasite’s cytoskeleton and protein–protein interactions of its components. The lysate is diluted to RIPA conditions for IP, and only the target protein (red) and covalently attached partners (green) are purified. (C) Western blot analysis of ILP1 denaturing IP recapitulates uncrosslinked (blue arrow) and crosslinked (black asterisks) ILP1 Azi mutants. As seen in the anti-Myc blot, the IP procedure eliminates the majority of the higher molecular weight background and reveals Myc-reactive crosslinked bands for residues T184, T187, and I188 (red arrowhead), demonstrating that IMC3 is indeed the partner at these residues. A small amount of uncrosslinked IMC3 that is likely to be reassociating with ILP1 is seen following dilution of the denatured lysate for IP (blue arrow). In contrast, no anti-Myc signal is seen for E209, indicating that this residue does not bind to IMC3. Azi, p-azidophenylalanine; HA, hemagglutinin; ILP1, IMC localizing protein 1; IP, immunoprecipitation.