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

Cells grown in six-well plates were washed with PBS and harvested in 100 µl lysis buffer (TBS, 1% Triton-X100, and protease inhibitors) or lysed in an alternative lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, protease inhibitors, and 5 mM sodium fluoride) for detection of phospho-ATG13. The lysates were incubated on ice for 30 min, vortexed, and centrifuged at 14,000 g for 10 min at 4°C. The supernatants were collected and mixed with Laemmli loading buffer (Laemmli, 1970) . Equal protein amounts were separated by SDS-PAGE, and after standard Western blotting, proteins were detected using specific antibodies and the Odyssey Imaging System (LI-COR Biosciences). Protein signal intensities (densitometric values) were normalized against a tubulin loading control for each sample. Densitometric values were determined and quantified on Western blots at nonsaturating exposures using the ImageJ software (Schneider et al., 2012) .