Rabbit anti bmi1

Immunoblotting was performed using the following antibodies: rabbit anti-Bmi-1 (#5856; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit anti-p16 (sc1207; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rat anti-p19Arf (sc32748; Santa Cruz Biotechnology, Inc.), rabbit anti-p15 (sc613; Santa Cruz Biotechnology, Inc.), rabbit anti-p18 (sc865; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-Rb (ser807/811) (#9308; Cell Signaling Technology, Inc.), rabbit anti-phospho-Akt (ser473) (#4060; Cell Signaling Technology, Inc.), rabbit anti-Akt (#4691; Cell Signaling Technology, Inc.), and mouse anti-β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA).

BCBL-1 cells were treated with DMSO, NaB+TPA, or 1μM Romidepsin or Panobinostat for 48 hours. BCBL-1 cells were treated with DMSO, NaB+TPA, or 10μM (+)-JQ1 or PTC-209 for 120 hours, and fresh compounds were added for an additional 48 hours. Cells were harvested, washed with PBS, and lysed in NP-40 lysis buffer (0.5% NP40, 150mM NaCl, 50mM Tris-HCL pH 8.0, 1x Complete Protease Inhibitor (Roche)). Protein concentration was determined by Bradford assay, and equivalent quantities of protein (15–30μg) were loaded per lane. Proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The following primary antibodies were used: mouse anti-KSHV ORF45 (2D4A5; Thermo Scientific), mouse anti-K8α (8C12G10G1; Santa Cruz Biotechnology), rabbit anti-Bmi1 (D20B7; Cell Signaling), and goat anti-actin, horseradish peroxidase (HRP)-conjugated (C-11; Santa Cruz). Supernatants from ATCC hybridoma PTA-2220 (v6m 12.1.1) [53 (link)] were used to probe for vIL-6.

Cells were lysed in ice-cold radio-immunoprecipitation (RIPA) buffer containing protease inhibitor and phosphatase inhibitor cocktail (Alfa Aesar, Stoughton, MA, USA). Protein was quantified using the Bradford Assay (BioRad, Hercules, CA, USA). Proteins (20–30 μg) were resolved in SDS-polyacrylamide gels (10%) and transferred to PVDF membranes using a Tris-glycine buffer system. Membranes were blocked with 5% non-fat dry milk in 0.1% Tween20 in TBS (TBST) for 1 h at room temperature followed by probing with primary antibodies and gently rocking overnight at 4 °C. Antibodies used for immunoblotting were Mouse anti-E-cadherin (Cell Signaling, Danvers, MA, USA), Rabbit anti-N-cadherin (Cell Signaling), Rabbit anti-vimentin (Protein Technologies, Tucson, AZ, USA), Rabbit anti-survivin (Cell Signaling), Rabbit anti-Lasp1 (Cell Signaling), Rabbit anti-BMI1 (Cell Signaling), and Mouse anti-β-actin (BD Biosciences, San Jose, CA, USA). The next day, the blots were washed with TBST three times, 5 min each time. They were incubated with secondary antibodies (1:2000) at room temperature for 1 h. Chemiluminescent signals were detected with ECL™ prime (Thermo Fisher Scientific, Waltham, MA, USA) using the Biorad ChemiDoc system. If necessary, blots were stripped with ECL Stripping Buffer (Li-Cor, Lincoln, NB, USA) following the manufacturer’s protocol. Bands were quantified using ImageJ.