Facs buffer

Vero cells were pretreated with inhibitors at the indicated concentrations, followed by infection with Ba71V or BPP30GFV at a moi of 1 pfu/cell. At 16 hpi, cells were washed with PBS and harvested by trypsinization. Flow cytometry experiments were performed by detection of infected cells with an anti-p72 monoclonal antibody followed by incubation with FITC-conjugated anti-mouse inmmunoglubulins 1:50, diluted in FACS buffer (Dako). When using the recombinant BPP30GFP green fluorescent the use of antibodies was not necessary. 10,000 cells per tube in triplicates were scored and analyzed in a FACS Canto II flow cytometer (BD Sciences, San Diego, CA, USA) to determine the percentage of infected cells per condition. Infected cell percentages obtained after inhibitors treatment were normalized to control values.

ITZ was purchased from Santa Cruz Biotechnology and 25-Hydroxycholesterol (25-HC) from Sigma Aldrich (St. Louis, MO, USA). Rabbit polyclonal anti-OSBP (Sigma) was used at 1:50 dilution, anti-PI4Kβ (Millipore, Burlington, MA, USA) at 1:300 dilution, anti-ACBD3 (Sigma-Aldrich) at 1:100 dilution and mouse monoclonal antibody to PDI (ab2792 abcam) at 1:1000; MVB were labeled with anti-CD63 antibody (Clone H5C6; Developmental Studies Hybridoma Bank, University of Iowa) at 1:200 dilution. MitoTracker CMXRos was obtained from Invitrogen (Waltham, MA, USA) and used at 100 nM. Other primary antibodies used were monoclonal antibodies against the virus major capsid protein p72 (Ingenasa, Madrid, Spain) used at a working dilution of 1:1000, anti-p30 antibody at 1:100 dilution (kindly given by Dr. J.M. Escribano, INIA) and swine anti-p54 antibody at 1:100. As secondary antibodies, we used anti-mouse immunoglobulin G (IgG) antibody conjugated to Alexa Fluor 594 and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA), both at 1:200 dilutions and FITC-conjugated anti-mouse inmmunoglubulins 1:50, diluted in FACS buffer (Dako, Agilent, Santa Clara, CA, USA).