GIPC3 AP-MS/MS from chick inner ear extracts

To understand how GIPC3 exerts its effects on cuticular plate dimensions, we examined the GIPC3 protein-interaction network in hair cells. We immunoaffinity purified GIPC3 from crosslinked chicken inner ear extracts that were enriched for stereocilia, but still contain large amounts of hair-cell cytoplasmic proteins (Morgan et al., 2016). We carried out two separate experiments, each with ~1000 chicken ears, where we stabilized protein complexes using primary amine-reactive homo-bifunctional N-hydroxysuccimide ester crosslinkers that are thiol-cleavable and hence reversible (Mattson et al., 1993). In one experiment, we used dithiobis(succinimidyl propionate) (DSP), a membrane-permeable crosslinker that crosslinks extracellular and intracellular complexes; in the other experiment, we used 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), which is membrane impermeant and thus only stabilizes extracellular and transmembrane complexes. We prepared soluble extracts of crude, crosslinked stereocilia and used these fractions for identifying GIPC3-interacting proteins.