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 were fixed with 4% PFA and washed and blocked with blocking buffer (PBS, 1% bovine serum albumin, and 0.1% saponin). Primary and secondary antibodies were diluted in the blocking buffer and incubated for 1 h. Nuclei were stained with Hoechst 33342 during the incubation with the secondary antibody for automated image acquisition. Alternatively, nuclei were stained with DAPI for confocal microscopy. Confocal microscopy was performed at room temperature using a laser-scanning microscope 700 (ZEI SS) with a 63× 1.4 DIC Plan-Apochromat oil-immersion objective. ZEN digital imaging software (ZEI SS) was used for image acquisition and processing of the images. All images were exported as TIFF images, and figures were finalized in Adobe Illustrator (Adobe).