Computational protocol: OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo

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Protocol publication

[…] LNCaP-FGC cells were metabolically labeled following the stable isotope labeling by amino acids in cell culture (SILAC) methodology []. Briefly, cell were cultured for at least 10 generations in RPMI medium (Biowest) supplemented with 10% dialyzed FBS (Sigma) and 28 and 48 mg/L of stable isotopic variants of arginine and lysine respectively (Cambridge isotope laboratories, Inc) as only source for these amino acids. Cells cultured with Arg10 (13C6; 15N4) and Lys8 (13C6; 15N2) were denominated “heavy”, “medium” when the amino acids were Arg6 (13C6;) and Lys4 (4,4,5,5-D4) and “light” with Arg0 and Lys0. All amino acids were purchased from Cambridge isotope laboratories, Inc. “Light” cells were transfected with siRNA control while “heavy” and “medium” cells were transfected with different siRNA targeting OTUB1. Whole cell extracts were purified and mixed in a 1:1:1 ratio and resolved by SDS-PAGE. After trypsin in-gel digestion, peptides were analyzed by LC-MS/MS using an orbytrap mass spectrometer. The obtained mass spectrometric raw data were analyzed in the MaxQuant environment [] with the integrated Andromeda searching engine and false discovery rate cut-off for peptide identification of 0.1 []. The effects of OTUB1 depletion were calculated as the result of dividing the normalized intensity of each “heavy” (siOTUB1_1) and “medium” (siOTUB1_2) protein by the intensity of the correspondent “light” (siControl) protein. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Neoplasms, Ovarian Neoplasms, Prostatic Neoplasms