Ankfy1

Rabbit polyclonal antibody to Becn1, Atg3, Atg7, and myc tag were from Cell Signaling Technology (Danvers, MA). Other antibodies were rabbit polyclonal to green fluorescent protein (GFP; Abcam, Cambridge, MA), mouse monoclonal to GFP (Takara, Mountain View, CA), rabbit polyclonal to LC3B (Sigma, St. Louis, MO), mouse monoclonal to EBOV glycoprotein (GP) clone 4F3 (IBT Bioservices, Gaithersburg, MD), mouse monoclonal to EEA1 (BD Biosciences, Franklin Lakes, NJ), and mouse monoclonal to GAPDH and rabbit polyclonal to Ankfy1 (Thermo Fisher, Waltham, MA).

siRNA-treated HeLa cells were collected 24 hours after the second siRNA transfection. Fifteen thousand cells were plated into wells of the 96-well plate in triplicate to be infected with EBOV-eGFP; the remaining cells were plated either into new 60-mm dishes (autophagy proteins) or into 8-chamber μ-slides (ibidi, Munich, Germany; Ankfy1) to be tested for gene silencing. After 24 hours, virus was added to the cells at a multiplicity of infection (MOI) of 0.1 for 24 hours. The cells were then fixed and nuclei stained with Hoechst 33342 dye. Infection efficiency was calculated as the ratio of infected cells (expressing eGFP) to cell nuclei. The effect of siRNA on gene expression was determined at the same time as infection. Autophagy protein depletion was assessed by immunoblotting. Depletion of Ankfy1 was verified by immunofluorescence. The cells grown on slides were fixed, permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with 5% goat serum (Thermo Fisher), stained with anti-Ankfy1 antibody overnight and then with Alexa Fluor 546–conjugated anti-rabbit secondary antibody and HCS CellMask blue stain (Thermo Fisher). Images were acquired using a Nikon Ti-Eclipse microscope (Nikon, Tokyo, Japan).