Bcl2 clone 124

Whole cell protein extracts were obtained by lysing 2–5 × 10⁶ cells with Laemmli sample buffer. Protein concentration was measured with the Micro BCA^TM Protein Assay Reagent kit (Thermo Scientific Pierce, Rockford, IL, USA). Protein extracts (25 μg) were subjected to reducing conditions before being subjected to electrophoresis on a polyacrylamide gel and then transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). One hour after blocking with 5% (w/v) non-fat milk in Tris-buffered saline with 0.1% Tween^®-20, the membranes were incubated with the specific primary antibodies against BCL-2 (clone 124, #M0887, Dako, Denmark), BIM (clone C34C5, #2933, Cell Signaling, Danvers, MA, USA), cleaved caspase 3 (#9661, Cell Signaling), ERK2 (clone 1B3B9, #05-157, Millipore, Temecula, CA, USA), NOXA (clone 114C307, #ab13654, Abcam, Cambridge, UK), MCL-1 (clone S-19, #sc-819, Santa Cruz Biotechnology, Dallas, TX, USA), PHB1 (clone H-80, #sc-28259, Santa Cruz Biotechnology), PHB2 (anti-REA, #07-234, Millipore), PARP (#9542, Cell Signaling), PUMA (#4976, Cell Signaling), β-Actin (clone AC-15, #A5441, Sigma-Aldrich) and Tubulin (clone Ab-1, #CP06, Oncogene, Darmstadt, Germany). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Amersham Place, Buckinghamshire, UK).

Immunohistochemistry (IHC) was performed using antibodies against MYC (clone EP121, Cell Marque, Rocklin, CA, USA), BCL2 (clone 124, DAKO, Carpinteria, CA, USA), BCL6 (clone LN22, Novocastra, Newcastle, United Kingdom), CD10 (clone 56C6, Novocastra), and MUM1 (clone Ma695, Novocastra). IHC staining was performed using the Ventana Benchmark XT system (Ventana Medical Systems, Tucson, AZ, USA) or a Bond-Max automated immunostainer (Leica Microsystems, Melbourne, Australia). Cell of origin was assessed according to the Hans criteria [11 (link)]. IHC score was determined to be the percentage of tumor cells with robust immunostaining evaluated by 10 % increments. Cutoff values (i.e. IHC scores) of ≥40 % for MYC, ≥30 and ≥60 % for BCL2 and ≥50 % for BCL6 were determined to have discriminant prognostic power based on the receiver operator characteristic (ROC) curves and were thus used for classifying cases into MYC-, BCL2- or BCL6-negative and-positive groups.