Ab90543

Cells were lysed in RIPA buffer containing complete mini protease inhibitor (Roche) for 15 min on ice with occasional vortexing. Samples were centrifuged, and cell lysates were mixed with SDS loading buffer and boiled at 95°C for 10 min. Sample were then loaded and separated on 10% SDS-PAGE gels, before transferring onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% milk diluted in PBS containing 0.05% Tween-20 (PBST) for 1 hour, before incubation with primary antibodies at 1:500-1000 dilution overnight at 4°C. Thereafter, membranes were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h before detection with Western Lightning Chemiluminescence (Perkin Elmer). Antibodies used included: anti-EBNA1 (sc-57719, Santa Cruz), anti-EBNA2 (ab90543, Abcam), anti-LMP1 (CS1-4, Dako), anti-LMP2A (MCA2467, Bio-rad), anti-GAPDH (mab374, Merck), anti-mouse IgG-HRP (ThermoFisher Scientific) and anti-rat IgG HRP (Santa Cruz).

Cell lysates were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA; Millipore) supplemented with 1× protease inhibitor cocktail (Thermo Scientific). Protein extracts were obtained by centrifugation at 3,000 × g for 10 min at 4°C. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay (Pierce). Lysates were boiled with 2× Laemmli sample buffer (Bio-Rad) containing 2.5% β-mercaptoethanol (Sigma-Aldrich). Proteins were resolved by gel electrophoresis on a 4 to 20% polyacrylamide gradient Mini-Protean TGX precast gel (Bio-Rad) and transferred to an Immobilon-P membrane (Millipore). Membranes were blocked in 5% milk in PBS-T for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against Lamin AC (Active Motif 39287), Lamin B1 (Abcam ab16048), EBNA2 (Abcam ab90543), LMP1 (Abcam ab78113), and actin (Sigma-Aldrich A2066) as recommended per the manufacturer. Membranes were washed, incubated for 1 h with the appropriate secondary antibody, either goat anti-rabbit IgG-HRP (Jackson Immuno Research) or rabbit anti-mouse IgG-HRP (Jackson Immuno Research). Membranes were then washed and detected by enhanced chemiluminescence.

Cells were washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer for 30 min on ice. After centrifugation at 4°C for 10 min, the supernatant was removed, and the protein concentration was estimated calorimetrically using a Bio-Rad detergent-compatible assay. Protein samples (30 μg) were loaded onto SDS-polyacrylamide gels at a percentage appropriate for electrophoretic separation. Antibodies used for Western blotting were as follows: antibodies against EBNA3A (ab16126; Abcam), EBNA3B (clone 6C9; Allday lab), EBNA3C (clone A10; a gift from M. Rowe, University of Birmingham), EBNA1 (a gift from P. Farrell, Imperial College), EBNA2 (ab90543; Abcam), EBNA-LP (JF-186) (82 (link)), LMP1 (CS1-4; Dako), γ-tubulin (T6557; Sigma), RBPJ (J7A11-161; a gift from B. Kempkes, Helmholtz Zentrum München), KDM2B (09-864; Millipore), BMI1 (05-637; Millipore), SUZ12 (sc-46264; Santa Cruz), and mCherry (Ab183628; Abcam). In all blots, γ-tubulin was used as a loading control. The appropriate horseradish peroxidase (HRP)-conjugated antibodies were used as secondary antibodies (all from GE Healthcare). An ECL kit (Amersham Biosciences) was then used for visualization by autoradiography. In some cases, the membrane used for Western blotting was cut horizontally after protein transfer in order to facilitate multiple antibody probes and a single loading control for each blot used.