Actin ab 5

The mouse anti-OROV antiserum (kindly donated by Dr. Luis Tadeu Moraes Figueiredo) was used for immunofluorescence, western blot, and immunoelectron microscopy assays. Rabbit polyclonal antibodies to Calnexin-2 (H-70; Santa Cruz, CA), Giantin (Covance, NJ) and Lamp-1 (Cell Signaling Technology, MA), sheep polyclonal antibody to TGN46 (AbD Serotec, Oxford), and J2 mouse monoclonal antibody (Mab) anti-double-stranded RNA (dsRNA; English & Scientific Consulting Kft, Hungary), were used for immunofluorescence assays. Polyclonal goat antibodies to Alix (N-20; Santa Cruz Biotechnology, CA), and mouse monoclonal antibodies to Tsg101 (BD Bioscience, CA), GAPDH (Sigma Aldrich, SG) and Actin (Ab-5; BD Bioscience, CA) were used for immunoblot experiments. Secondary antibodies conjugated to Alexa-fluorophores as indicated in the figure legends were purchased from Thermo Scientific (Rockford, IL). Horseradish peroxidase-conjugated donkey anti-mouse immunoglobulin G (IgG), donkey anti-rabbit IgG, and donkey anti-goat IgG were obtained from GE Healthcare.

Cell lysates were prepared as described previously (11 (link)) and analyzed using 4% to 12% or 10% NuPAGE gels resolved in MES (morpholineethanesulfonic acid) running buffer (Thermo Fisher Scientific). Membranes were stained using the following antibodies: LC3B, clone 5F10 (Nanotools; 0231-100); p62/SQSTM1 (BD Bioscience; 610832); phospho-S6 Ser240/Ser244 (Cell Signaling; 2215); total S6 (54D2) mouse monoclonal antibody (MAb) (Cell Signaling; 2317); phospho-ACC Ser79 (Cell Signaling; 3661); total ACC (Cell Signaling; 3662); phospho-ULK1 Ser757 (Cell Signaling; 6888); phospho-ULK1 Ser555-D1H4 (Cell Signaling; 5869); phospho-ULK1 Ser317-D2B6Y (Cell Signaling; 12753); total ULK1-D8H5 (Cell Signaling; 8054); and actin, Ab-5 (BD Bioscience; 612656). Detection was via anti-mouse or anti-rabbit Dylight-coupled secondary antibodies and LiCor Odyssey infrared scanning.