Odyssey infrared system

Western blot analysis of experimental and control cell lysates was carried out using the Odyssey^® Infrared System (LI-COR Biosciences, Lincoln, NE) and polyclonal anti-phospho-AMPK-α (Thr 172) antibody (Cat # 07-681SP), polyclonal Anti-p21 antibody (Cat # 0506550), and polyclonal Anti-Kip1 (p27) (Cat # 060445) were purchased from Millipore (Millipore Corporation, Iselin, NJ ). Polyclonal beta actin antibody (Cat # sc-1615) and polyclonal RASSF1C antibody (sc-18724) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA), and fluorescently-labeled secondary antibodies IRDye^® 680 and 780 RD Infrared Dye were purchased from LI-COR (LI-COR Biosciences, Lincoln, NE). The experiments were repeated at least 3 times. Protein levels were normalized to actin levels (the loading control).

SV589 cells seeded in 24-well plates were cultured overnight in Medium C containing 150 μM E64. PCSK9 labeled with thiol-cleavable biotin was incubated with IRDye800CW-labeled streptavidin for 1 h at 37°C, and the complexes were then added to the cells. After 1 h, the cells were incubated with 20 mM Tris(2-carboxyethyl)phosphine (TCEP) in buffer B (PBS, 0.1 mM CaCl₂, 2 mM MgCl₂, 0.5% BSA (w/v)) for 20 min at 4°C, then washed twice for 10 min with 5 mg/ml iodoacetamide in buffer B. The cells were washed again with buffer B and PBS-CM (PBS, 0.1 mM CaCl₂, 2 mM MgCl₂), then incubated in Medium A, supplemented with TCEP and E64, for 6 h at 37°C. The plate was directly scanned on the LI-COR Odyssey infrared system at time intervals. Signal intensity was quantified using Odyssey 2.0 software, background fluorescence of untreated wells was subtracted, and signal normalized to DNA levels stained by DRAQ5 emitting at a separate wavelength (Cedarlane Laboratories, Canada) (1:10,000).

BHK-21 cells were electroporated with RuV RNAs, and then seeded in four 35 cm wells and cultured in growth media for 72 h. Culture medium was harvested at the indicated times and infectious particles titered by infectious center assay, all as described above. The efficiency of particle assembly was determined at 48 h post-electroporation by pelleting half of the culture medium at 210,000× g for 3 h at 4°C and lysing the cells. Proteins from both samples were resolved by SDS-PAGE and analyzed by Western-blot using an anti-RuV serum (Meridian Life Science). Virus particle release was evaluated by comparing the ratio of the virus and cell lysate E1, E2 and capsid bands using the Odyssey Infrared system (Li-Cor).

Western blot analysis was carried out using the Odyssey Infrared System (LI-COR Biosciences, Lincoln, NE) and anti-β-catenin antibody #9582 (Cell Signaling, Danvers, MA), anti-PIWIL1 antibody ab12337 (Abcam, Cambridge, MA), anti-HA antibody clone 16B12 (Covance, Berkeley, CA), monoclonal beta actin antibody AC-74 (Sigma, St. Louis, MO), and fluorescently-labeled secondary antibodies IRDye 680RD Infrared Dye (LI-COR Biosciences, Lincoln, NE). The experiments were repeated at least 3 times. Protein levels were normalized to actin levels (the loading control) with standard deviations.