Stable Isotope Labeling by Amino-Acid (SILAC) Analysis

LNCaP and LNCaP-JAK1 cells were grown in RPMI devoid of lysine and arginine (Thermo, cat. #A2494401), supplemented with 10% dialyzed FCS (Biological Industries, cat. #04-011-1A) and antibiotics for 10 cell divisions (~3 weeks). “Heavy” culture medium was supplemented with ¹³C₆¹⁵N₂-lysine (146 mg/mL, cat. # CNLM-291-H) and ¹³C₆¹⁵N₄-arginine (84 mg/mL cat. #CNLM-539-H) both from Cambridge Isotope Laboratories. “Light” labeled culture medium was supplemented with unmodified lysine and arginine at the same concentrations. To avoid potential bias in the analysis, in any given condition, cultures were labeled with heavy (H) or light (L) amino acids, or with the reciprocal labeling. Each H–L pair was repeated twice. Thus, quantification of each condition was based on four independent replicates. Labeled cells, either treated or untreated, were trypsinized, counted, and washed twice in cold PBS. Samples were digested by trypsin and analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) on Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The data were analyzed and quantified with MaxQuant 1.5.2.8 (54), using the human Uniprot database. Proteins were identified with false discovery rate (FDR) < 0.01. Proteins that exhibited differential expression (|log₂ ratio| ≥ 0.5) in at least three out of four replicates, in any given condition, were chosen for further comparisons. Proteomic analysis of the samples was done at the biomedical core facility at the genomic center (Technion, Israel).