Anti acbd3

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).

The rabbit antiserum and the mouse monoclonal antibody against CVB3 3A were described previously (18 (link), 26 (link)). Mouse monoclonal antibodies included anti-ACBD3 (Sigma), anti-myc (Sigma), anti-GM130 (BD Biosciences), and antigiantin (Enzo Life Science). Rabbit polyclonal antibodies included anti-PI4KB (Millipore), anti-myc (Thermo Fisher Scientific), anti-TGN46 (Novus Biologicals), anticalreticulin (Sigma), anti-FLAG (Sigma), and anti-EGFP (a gift from J. Fransen, NCMLS, Nijmegen, The Netherlands). Goat anti-rabbit and goat anti-mouse antibodies conjugated to Alexa Fluor 488, 596, or 647 (Molecular Probes) were used as secondary antibodies for immunofluorescence analysis. For Western blot analysis, IRDye goat anti-mouse or anti-rabbit (LI-COR) were used.

Proteins were fractionated by 12% or 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes by the wet method. PVDF membranes were blocked with Tris-buffered saline-0.1% Tween 20 (vol/vol) that contained 5% nonfat dry milk, and were probed with antibodies indicated. Primary antibodies were used at the following dilutions: anti-SCAMP3 rabbit polyclonal, 1:4,000 (GeneTex); anti-FLAG mouse monoclonal, 1:4,000 (Sigma); anti-3D mouse monoclonal, 1:4,000 (a gift from Shin-Ru Shih, Chang Gung University); anti-3A rabbit polyclonal, 1:1,000 (a gift from Jim-Tong Horng, Chang Gung University); anti-PI4KIIIβ, 1:1,000 (Merck Millipore); anti-calnexin 1:500 (Santa Cruz Biotechnology); anti-NS3, 1:5,000 (GeneTex); anti-ACBD3, 1:1,000 (Sigma-Aldrich); and anti-actin, 1:5,000 (Millipore).