Las af version 1

Cells were fixed with 3.7% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min and then incubated for 1 h with mouse anti-V5 tag mAb (Life Technologies) followed by decoration with goat anti-mouse IgG fluorescein-conjugated (ICN Cappel Inc), and with rabbit anti-HA policlonal antibody (Sigma-Aldrich) followed by decoration with goat anti-rabbit Alexa-fluor 594 (Life-technologies). RedDot^™2 far-red nuclear stain (Biotium, Inc., Hayward) diluted 1:400 was utilized as a nuclear marker. For p53 protein detection, rabbit anti-p53 polyclonal Ab (Santa Cruz Biotechnology) followed by goat anti-rabbit Alexa-fluor 594 (Life-technologies) was used. Control incubations demonstrated no cross-reactivity between the anti-immunoglobulin conjugates or between the anti-immunoglobulin conjugate and irrelevant primary antibodies. All samples were examined using a confocal microscope Leica TCS SP5 and processed with LAS AF version 1.6.3 software (Leica Microsystems). To prevent cross emission spectra, specific lasers (488 nm, 546 nm and 633 nm) were activated in sequential mode to acquire the images.

A total of 4 × 10⁴ HEK-293T cells was seeded on chamber glass slides (BD Biosciences, San Diego, CA, USA) and transfected with vectors expressing either Nef^mut, Nef^mut/E7, or GC-AG Nef^mut/E7. Forty-eight hours later, cells were permeabilized through the Cytofix-Cytoperm-based protocol (BD Biosciences, San Diego, CA, USA) and then labeled with 1:2000 diluted anti-Nef mAb MATG020 (kindly provided by O. Schwartz, Paris, France), followed by incubation with 1:2500 diluted Alexa 488-conjugated goat anti-mouse (Invitrogen). Coverslips were mounted using an anti-fade mounting medium containing 4’-6-diamidino-2-phenylindole (DAPI). Images were acquired using a Leica TCS SP5 confocal microscope and analyzed by the LAS AF version 1.6.3 software (Leica microsystems, Wetzlar, Germany).