An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A.; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

Immunoprecipitation and protein mass spectrometry HA immunoprecipitation followed by mass spectrometric analysis was performed as previously described (Behrends et al., 2010) . In brief, 293T-REx cells expressing HA-tagged proteins for 24 h were infected with CV for 7 h at an MOI of 2 or left uninfected before they were harvested and frozen at −80°C. Subsequently, cells were thawed and lysed with ice-cold MCLB buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, and complete EDTA-free protease inhibitor tablets; Roche), cleared through 0.45-µm spin filters (EMD Millipore), and immunoprecipitated using anti-HA-agarose (Sigma-Aldrich) overnight. After intensive washing, proteins were eluted with HA peptide (250 µg/ml; Sigma-Aldrich) and precipitated with TCA (Sigma-Aldrich), followed by digestion with trypsin (Promega) and desalting by custom-made stage tips. Samples were analyzed in technical duplicates on a LTQ Velos (Thermo Fisher Scientific), and spectra were identified as previously described (Huttlin et al., 2010) . For CompPASS analysis, we used 142 unrelated bait proteins that were all previously processed in the same way (Sowa et al., 2009; Behrends et al., 2010) . Weighted and normalized D-scores (WD N -scores) were calculated based on average peptide spectral matches (APSMs). Proteins with WD N ≥ 1 and APSM ≥ 2 were considered as new interacting proteins.