C9orf72 ubiquitin and interaction proteomics

Version 1

C9orf72 binds SMCR8 to from a robust complex that regulates small GTPases, lysosomal integrity and autophagy. In contrast to this functional understanding, we know far less about assembly and turnover of the C9orf72-SMCR8 complex. Loss of either subunit causes the concurrent ablation of the respective partner. However, the molecular mechanism underlying this interdependence remains elusive.

ollowing HA-immunoprecipitation of C9orf72 from SMCR8 KO cells, proteins were precipitated with 20% TCA and incubated for 20 min on ice. After centrifugation at 20,000 x g, 4°C for 30 min, the supernatant was discarded, 10% cold TCA was added to pellets and centrifuged again. Pellets were washed 3x in cold acetone, centrifuged and then dried in a speed vacuum concentrator. For in-solution tryptic digestion, pellets were resolved in 50 mM ammonium bicarbonate (ABC) pH 8.0 with 10% acetonitrile ...