Ab36623

H1299 cells were grown, fixed and permeabilized as described for immunofluorescence. Samples were overnight hybridized with 50 ng of a Globin DNA probe (5′-GGCCTCACCACCAACTTCATCCACGTTCACCTTGCAAAAA-3′) conjugated to digoxigenin in the 3′ end. Afterwards, samples were saturated with PBS 3% BSA, 0.1% saponine and incubated for 2 h with mouse monoclonal anti-digoxigenin (DI-22, Sigma) and rabbit polyclonal anti-NCL (ab22758, Abcam) primary antibodies diluted in blocking solution. The proximity ligation assay (PLA) was carried out using the Duolink PLA in situ kit (Sigma) following the manufacturer's protocol. For co-staining of HIV-1 Rev protein in PLA samples, chicken polyclonal anti-HIV-1 Rev antibody (ab36623, Abcam) and goat anti-chicken IgY conjugated to Alexa Fluor^® 568 (Abcam) were added to primary antibodies and Duolink secondary antibodies mixes, respectively. For co-staining of NCLΔNLS or nucleolus, samples were incubated for 45 min with mouse monoclonal anti-HA-Tag antibody conjugated to Alexa Fluor^® 488 (6E2, Cell Signaling) or anti-Fibrillarin antibody conjugated to Alexa Fluor^® 488 (EPR10823B, Abcam) after the PLA amplification step.

Whole cell lysates were prepared 48 h post-transfection and protein concentration was measured using a Bradford assay. Samples were electrophoretically separated in NuPAGE^® Bis-Tris gels 10% (Invitrogen), transferred onto 0.45 μm nitrocellulose membranes (GE) and blotted under standard conditions using the following antibodies: mouse monoclonal anti-HBB antibody (2H3, Sigma), rabbit polyclonal anti-OVA antibody (C6534 Sigma), mouse monoclonal anti-actin (AC-15 Sigma), mouse polyclonal anti-HA antibody (a kind gift from Borek Vojtesek, Masaryk Memorial Cancer Institute, Brno, Czech Republic) and chicken polyclonal anti-HIV-1 Rev (ab36623, Abcam). Anti-mouse (Dako), anti-rabbit (Dako) or anti-chicken (Sigma) secondary antibodies conjugated to horseradish peroxidase were used to generate immunocomplexes revealed with enhanced chemiluminescence (Thermo Scientific). Membranes were scanned in a MyECL imager (Thermo Scientific) and signal intensity was determined using My Image software (Thermo Scientific).

H1299 cells were plated on 12-mm-diameter coverslips in 24-well plates, and transfected with the indicated constructs or pCDNA3. At 24 h post-transfections cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with PBS 0.4% Triton X-100, 0.05% CHAPS for 10 min at room temperature and saturated with PBS 3% BSA for 30 min. Samples were incubated overnight at 4°C with rabbit polyclonal antibody anti-NCL (ab22758, Abcam) or mouse monoclonal anti-HA-Tag conjugated to Alexa Fluor^® 488 (6E2, Cell Signaling). A goat anti-rabbit Ig antibody conjugated to Alexa Fluor^® 647 (Sigma) was used as secondary antibody when necessary. RNA-FISH assays were performed employing Globin and OVA probes obtained from Biosearch Technologies according to the manufacturer's protocol. For nucleolar co-staining, samples were incubated with anti-nucleolin conjugated to Alexa Fluor^® 488 (ab154028, Abcam) after probes hybridization. For immunofluorescence coupled to RNA-FISH, samples were incubated with chicken polyclonal anti-HIV-1 Rev antibody (ab36623, Abcam) after hybridization and immunocomplexes were detected under standard conditions using goat anti-chicken IgY conjugated to Alexa Fluor^® 568 (Abcam). Samples were examined in an LSM 800 confocal laser microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany).