Bioruptor equipment

After knockdown experiments, cells were lysed in Triton‐X lysis buffer containing 50 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X‐100, 1 mM PMSF, 1 mM DTT, and 1× Halt protease inhibitor cocktail (Thermo Fisher Scientific), after which the lysates were sonicated four times for 30 s at medium power with Bioruptor equipment (Diagenode Inc.), and cellular debris was removed by centrifugation. Samples were resuspended in 2× Laemmli sample buffer and heated at 95°C for 5 min. Proteins were separated by Mini‐PROTEAN TGX Precast Gels (Bio‐Rad), and immobilized onto PVDF membranes (Immobilon‐P, Millipore, Merck). Primary antibodies against PIM1 (1:2000, Abcam, ab224772), PIM2 (1:2000, OriGene, TA501166), PIM3 (1:1000, OriGene, TA351349), ERG (1:5000, EPR3864; Epitomics), β Tubulin (1:40 000, Sigma‐Aldrich), or Fibrillarin (1:1000, Cell Signaling Technology) were used together with anti‐mouse HRP‐conjugated antibody produced in rabbit (1:10 000; DAKO) or anti‐rabbit HRP‐conjugated antibody produced in swine (1:5000; DAKO). Chemiluminescence reactions were generated using either Amersham^TM ECL Plus or ECL Prime reagents (GE Healthcare Life Sciences).

LNCaP cells (ATCC) were cultured under the recommended conditions. Cells and sections of frozen tissue were lysed in Triton-X lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0,5% Triton x-100, 1 mM PMSF, 1 mM DTT and 1× complete protease inhibitor cocktail (Roche Inc., Mannheim, Germany), after which the lysates were sonicated four times for 30 s at medium power with Bioruptor equipment (Diagenode Inc., Liège, Belgium), and cellular debris was removed by centrifugation. Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Immobilon-P; Millipore Inc., Billerica, Massachusetts, USA). Primary antibodies against ACO2 (HPA001097; Sigma-Aldrich, St. Louis, MO, U.S.A.; dilution 1:1000), MDH2 (HPA019714; Sigma-Aldrich; 1:1000), and pan-actin (ACTN05; NeoMarkers, Portsmouth, NH, USA; 1:1000) were used and detected using anti-rabbit HRP-conjugated antibody produced in swine (1:5000, DAKO Inc., Denmark) or by anti-mouse HRP-conjugated antibody produced in rabbit (1:5000, DAKO Inc., Denmark) and Western blotting luminol reagent (Santa Cruz Inc., Santa Cruz, California, USA) with autoradiography. Original scans including molecular weight information for the western blots are presented in Supplementary Fig. 19.