For Western blotting, cells were lysed with 1% SDS. 10 μg cell lysate was separated by SDS-PAGE using 12.5% tris-glycine SDS-PAGE gels and transferred to a PVDF membrane. Membranes were probed with anti-YB-1 antibody (EP2708Y, 1:100000 or EP2706Y, 1:10 000, Abcam, Cambridge, UK), anti-β-actin antibody (AC15-A544, 1:50000, Sigma, Bornem, Belgium) or anti-LEDGF/p75 antibody (A300-847A, 1:500, ImTec, Antwerpen, Belgium). Detection was done with chemiluminescence using ECL (Pierce, ThermoScientific, Erembodegem, Belgium) after probing the blots with horseradish peroxidase coupled secondary antibody (1:1000–1:10 000, DAKO, Agilent technologies, Diegem, Belgium). Protein concentrations were determined using the Pierce bicinchoninic acid assay (BCA, Thermo Scientific, Erembodegem, Belgium).
For immunocytochemistry (ICC), 3 x 104 HeLaP4 or 6 x 105 SupT1 cells were seeded on PolyD-lysine coated chamber slides (Nunc, Roskilde, Denmark) and fixed with 2% paraformaldehyde 24 hours post seeding. YB-1 expression was detected with anti-YB-1 antibody (Abcam EP2708Y, 1:2000), followed by incubation with a Alexa488-conjugated secondary antibody (Life Technologies, ThermoScientific, Gent, Belgium) and DNA was stained with DAPI (1 μg/mL).
For immunocytochemistry (ICC), 3 x 104 HeLaP4 or 6 x 105 SupT1 cells were seeded on PolyD-lysine coated chamber slides (Nunc, Roskilde, Denmark) and fixed with 2% paraformaldehyde 24 hours post seeding. YB-1 expression was detected with anti-YB-1 antibody (Abcam EP2708Y, 1:2000), followed by incubation with a Alexa488-conjugated secondary antibody (Life Technologies, ThermoScientific, Gent, Belgium) and DNA was stained with DAPI (1 μg/mL).