FIGURE 5.

Isolation of HFY-NL2-associated proteins. Brain lysate from adult Thy1-HFY-NL2 mice was subjected to two rounds of affinity purification. Following the first step of binding to a Ni-NTA column via the His₆ tag, the purified material (Ni eluate) was immunoprecipitated with biotinylated anti-GFP antibody and streptavidin-agarose via the YFP tag resulting in doubly purified material (Anti-GFP IP). A, SDS-PAGE and SYPRO Ruby stain shows a reduction in total amount and complexity of material during the two-step purification. B, Western blot shows increasing enrichment of the HFY-NL2 transgene (black arrow) as well as co-purification of some endogenous NL2 (gray arrow) and gephyrin.