Computational protocol: Rab7A Is Required for Efficient Production of Infectious HIV-1

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[…] Cells were lysed for 30 min at 4°C using lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 0.1% (v/v) sodium deoxycholate), supplemented with complete protease inhibitor cocktail (Roche Diagnostics). Cell lysates were spun for 10 min at 20,000 g and supernatants were recovered and mixed with 2x Laemmli sample buffer, (Sigma). Samples were boiled and proteins resolved on 12% SDS–PAGE gels. Proteins were transferred onto Hydrophobic polyvinylidene difluoride (PVDF) membranes and incubated in blocking solution (TBS–0.05% (v/v) Tween 20 supplemented with 5% (w/v) milk) for 30 min. Blots were rinsed with TBS containing 0.05% (v/v) Tween 20 and incubated with primary antibody in the blocking solution overnight. The membranes were rinsed as before and incubated for 30 min with the appropriate HRP-conjugated secondary antibody diluted in the blocking solution. The membranes were rinsed again and immunodetections were performed using the enhanced chemiluminescence ECL substrate (GE healthcare Europe GMB, France).For Env quantification, the membranes were incubated with anti-SUgp120 (110H) and TMgp41 (41A), washed and incubated with Dylight-800 Goat Anti-mouse IgG antibody (KPL, Gaithersburg, USA). The corresponding signals were then acquired with the Odyssey Infrared Imaging System (Li-Cor) for further quantification using ImageJ software. [...] For intracellular staining of CA and Env, cells were grown on cover slips, fixed with 4% (w/v) paraformaldehyde (PFA) in PBS for 20 min, permeabilized and blocked with 0.2% (w/v) BSA/0.1% (w/v) saponin in PBS (blocking solution) for 30 min. Cells were then incubated for 30 min with mouse anti-CAp24 (25A, Hybridolab) and human anti-Env HIV-1 gp120 (2G12, National Institute for Biological Standards and Control Centralised Facility for AIDS Reagents, NIBSC) in blocking solution. Cells were washed and incubated for 30 min with Alexa 488-conjugated donkey anti-mouse and Alexa 594-conjugated goat anti-human antibodies (Invitrogen).For extracellular staining of Env, living cells were incubated for 1 h at 4°C with human anti-Env HIV-1 gp120 antibody (2G12). Cells were then labelled with Alexa 594-conjugated goat anti-human antibody, washed, fixed with 4% PFA in PBS for 20 min, permeabilized and blocked for 30 min in blocking solution. Cells were then incubated for 30 min with mouse anti-MAp17 (18A, Hybridolab) in blocking solution as described above, washed and labelled for 30 min with Alexa 488-conjugated donkey anti-mouse antibody.For intracellular staining of BST2, cells grown on cover slips were permeabilized in PBS/0.1% BSA/0.05% saponin before fixation with 4% PFA in PBS for 15 min . PFA-fixed cells were permeabilized and blocked for 30 min, and incubated for 30 min with mouse polyclonal anti-BST2 (Abnova, Germany) alone or along with anti-Env HIV-1 gp120 antibody (2G12). Cells were then washed and incubated for 30 min with appropriate fluorophore-conjugated secondary antibodies.For colabelling of BST2 with CD63, HeLa cells transfected with either siRNA control Luciferase (siLuc) or siRNA targeting Rab7A were labelled with mouse polyclonal anti-BST2 antibody followed by an Alexa 594-conjugated donkey anti-mouse antibody. Cells were then incubated in blocking solution containing mouse serum (10%) for 20 min before immunolabelled with mouse anti-LAMP-3(CD63)-FITC (MX-49.129.5, Santa Cruz).For extracellular staining of BST2, living cells were incubated for 1 h at 4°C with mouse monoclonal anti-BST2 (Abnova) alone or together with human anti-Env HIV-1 gp120 (2G12). Then, cells were labelled with appropriate fluorophore-conjugated secondary antibodies, washed, fixed with 4% PFA in PBS for 20 min, permeabilized and blocked for 30 min. For MAp17/BST2 colabelling, living cells were incubated for 1 h at 4°C with rabbit polyclonal anti-BST2 (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID), then labelled with appropriate fluorophore-conjugated secondary antibodies, washed, fixed with 4% PFA in PBS for 20 min, permeabilized and blocked for 30 min. Cells were then incubated for 30 min with mouse anti-MAp17 (18A, Hybridolab) in blocking solution as described above.Stained cells were analyzed with a Leica DMI 6000 microscope or Leica Spinning Disk confocal microscope. Series of optical sections were recorded and image processing was performed using ImageJ and Adobe Photoshop CS2 software. Quantitative Env/BST2 and CD63/BST2 colocalization analysis was done with JACoP tool (ImageJ software) using Pearson's correlation coefficient . The estimation of Pearson’s correlation coefficient is one of the standard techniques applied for matching one image to another in order to describe the degree of overlap between two channels, taking into account the environment of each pixel. A value of +1 is the result of complete co-localization between two channels. The Pearson coefficient was calculated for twenty images per condition, with about 10 cells per image. Bars represent the mean ± SD from each image. […]

Pipeline specifications

Software tools ImageJ, Coloc, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Human immunodeficiency virus 1
Diseases HIV Infections