Rabbit anti flag polyclonal antibody

Human 293T cells were seeded at 8 × 10⁵ cells per well in six-well plates and transfections were carried out using the PEI method. To assay for Vif-mediated degradation of A3G, the following plasmids and amounts were used: 0.34 and 0.67 μg of pFlag-A3G, 2.5 μg of Vif, and 0.2 μg pGL (used as a positive control for transfection). To maintain equivalent DNA amount, pcDNA3.1 was added as needed. After 48 h, cell lysates were harvested and immunoblotting analyses were performed to detect steady-state expression levels of A3G in the absence or presence of Vif. Flag-A3G was detected using a rabbit anti-Flag polyclonal antibody (Sigma). HIV-1 Vif, endogenous CBFβ and tubulin were probed as described above.

Immunocytochemistry was performed as previously described [9 (link)] except that mouse anti-HA.11 monoclonal antibody (Covance Research Products, Princeton, NJ, USA) and rabbit anti-FLAG polyclonal antibody (Sigma Aldrich Ltd, St Louis, MO, USA) were diluted 1:5,000 in PBS. Cells were then washed with PBS for 3x 5 minute intervals prior to incubation with 4 μg/mL goat anti-mouse FITC and 2 μg/mL goat anti-rabbit Alexa 568 or 647 secondary antibodies (Invitrogen, Carlsbad, CA, USA) diluted in PBS + 0.1% BSA, for two hours at room temperature. Following this, cells were washed with PBS for 3x five minute intervals. Nuclei were counter-stained with 600 nM DAPI (Invitrogen, Carlsbad, CA, USA) following staining with secondary antibody. Slides were prepared for confocal microscopy as previously described [3 (link)]. For live cell imaging, the culture media was removed 48 hours post-transfection and the cells were washed 1x with warm Hanks Balanced Salt Solution (HBSS) (Invitrogen) + 1 g/L glucose + 25 mM HEPES. Cells were then maintained in HBSS + 1 g/L glucose + 25 mM HEPES for live cell imaging.

Cultured cells were lysed in a buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% sodium deoxycholate, and protease inhibitor cocktail (Sigma-Aldrich). A Western blot analysis was then performed using a rabbit anti-FLAG polyclonal antibody (Sigma-Aldrich), or a mouse anti-GAPDH monoclonal antibody (clone 6C5; Abcam, Cambridge, UK). Immunoreactivity was visualized using a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA) and an ECL chemiluminescence system (GE Healthcare Bio-Science, Piscataway, NJ, USA). Whole blots, molecular markers, and densitometric intensity ratios are shown in Supplementary Figure S5.

Flag Co-IP assays were carried out as previously described [19 (link), 25 (link)]. Briefly, 293T cells were seeded at 4 × 10⁶ cells per 10-cm dish and transfected the next day by the polyethylenimine PEI method [19 (link), 32 (link)]. The following DNA amounts were used to transfect 293T cells: 4 μg Flag-CBFβ, 4 μg Vif, and 1.8 μg pGreen Lantern-1 (pGL) (GIBCO; control for transfections) expression plasmids. To maintain equivalent DNA amounts, pcDNA3.1 was used when needed. After 48 h, total cell lysates were harvested in 1 mL of lysis buffer and Co-IP was performed as previously described [19 (link), 25 (link)]. To detect eluted complexes as well as the input cell lysates, western blotting was performed. CBFβ was detected using a rabbit anti-Flag polyclonal antibody (Sigma) at a 1:2000 dilution, Vif was detected using a mouse anti-Vif monoclonal antibody [Clone 319] (ab66643; Abcam) at a 1:2000 dilution, and tubulin as loading control was detected using mouse anti-tubulin antibody (Sigma) at a 1:20,000 dilution. Rabbit and mouse primary antibodies were detected using 1:5000 dilutions of an IRDye^® 800CW-labeled goat anti-rabbit secondary antibody (Licor) or an IRDye^® 680-labeled goat anti-mouse secondary antibody (Licor). Protein bands were visualized and quantified using an Odyssey^® Infrared Imaging System (Licor).

The following antibodies were used in immunofluorescence, Western blot or immunoprecipitation analyses: anti-HA monoclonal antibody (mAb; Cell Signaling, #3724), anti-Flag rabbit polyclonal antibody (Sigma, #F7425), anti-V5 mAb (Invitrogen, #R960), anti-β-actin mAb (Santa Cruz Biotechnology, #sc-47,778), and a polyclonal serum against the IBDV VP3 protein (Fernandez-Arias et al., 1998 (link)). In addition, anti-chTRIM25 antibodies were generated for this study by immunizing animals with a synthetic peptide (produced at ProteoGenix, France). A polyclonal serum anti-muNS protein (kindly provided by Dr. José Manuel Martínez Costas, CIQUS, Universidad de Santiago de Compostela. Spain) has been previously described (Touris-Otero et al., 2004 (link)).

Transfected HEK293T or HeLa cells were harvested from 100 mm dishes 48 h post-transfection and lysed in IP buffer (50 mM Tris-HCl pH 7.5, 150–300 mM NaCl, 1mM Dithiotreitol (DTT) and 0.5% Nonidet P-40) plus protease inhibitors (Roche Molecular Biochemicals). For immunoprecipitation of the transiently expressed Flag-tagged proteins, cell extracts were incubated with 30 μl anti-Flag M2 affinity gel (Sigma) for 4 h at 4°C and the immunopurified proteins were analyzed by western blotting. Antibodies used for other immunoprecipitations or western blotting analysis were anti-Flag rabbit polyclonal antibody (Sigma), anti-Myc (9E10) mAb, anti-human MIZ-1 goat polyclonal antibody (R & D Systems), anti-EBNA3A sheep polyclonal antibody (Exalpha biologicals Inc), anti-Nucleophosmin mAb (Invitrogen) and anti-α-tubulin mAb (B-5–1–2, Sigma). The appropriate anti-mouse (GE Healthcare), anti-rabbit (GE Healthcare), anti-goat (Santa Cruz Biotechnology, Inc) or anti-sheep (Abcam) horseradish peroxidase (HRP)-conjugated antibodies were used as secondary antibodies. Myc-tagged proteins were also revealed using Myc (9E10) HRP-conjugated antibody (Santa Cruz Biotechnology, Inc). Western blots were revealed using Enhanced Chemiluminescence (ECL) (Pierce).