13c6 15n2 l lysine

U2OS cells were cultured under standard conditions in DMEM for SILAC supplemented with 10% dialysed FBS and L-proline, Pro0 (200 μg/ml; Sigma) to prevent conversion of arginine to proline. Differentially labelled amino acids were added to the media to generate SILAC media for three conditions: light (L-lysine, Lys0; L-arginine, Arg0; Sigma), medium (L-lysine-²H₄, Lys4; L-arginine-¹³C₆, Arg6; Sigma) and heavy (L-lysine-¹³C₆-¹⁵N₂, Lys8; L-arginine-¹³C₆-¹⁵N₄, Arg10; Sigma). L-lysine was supplemented at a final concentration of 146 μg/ml whereas L-arginine was supplemented at a final concentration of 84 μg/ml. Cells were cultured for a minimum of six passages and analysed by MS to confirm amino acid incorporation before experiments proceeded.

Flp-In 293 T-REx cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium high glucose with 10% (v/v) fetal bovine serum, 2 mM L-glutamine. Cell lines stably expressing FLAG/HA-tagged RC3H1 protein were generated by co-transfection of pFRT/TO/FLAG/HA constructs with pOG44 (Invitrogen). Cells were selected by adding 15 μg ml⁻¹ blasticidin and 100 μg ml⁻¹ hygromycin (Invivogen). Expression of epitope-tagged proteins was induced by addition of 1 μg ml⁻¹ doxycyclin. The expression of FLAG/HA-tagged RC3H1 protein was assessed by western analysis using mouse anti-HA.11 monoclonal antibody (Covance). For quantitative proteomics, cells were grown in SILAC medium as described before30 (link)45 (link). Briefly, Dulbecco's modified Eagle's medium GlutaMAX lacking arginine and lysine (PAA) supplemented with 10% dialysed fetal bovine serum (Gibco) was used. Amino acids (84 mg l^{−1 13}C₆¹⁵N₄L-arginine plus 146 mg l^{−1 13}C₆¹⁵N₂L-lysine or 84 mg l^{-1 13}C₆-L-arginine plus 146 mg l⁻¹ D4-L-lysine) or the corresponding non-labelled amino acids (Sigma) were added to obtain ‘heavy' ‘medium' or ‘light' cell culture medium, respectively. Labelled amino acids were purchased from Sigma Isotec.

The human hepatic HuH7 cells (HuH-7) and human embryonic kidney 293 cells (HEK-293) were originally acquired from ATCC and the human colorectal cancer 116 cells (HCT-116) were obtained from the Japanese Collection of Research Bioresources Cell Bank. HuH-7, HEK-293 and HCT-116 were individually grown at 37 °C in a 5% CO₂ humidified incubator (to note, all three cell lines used in this study were found to be mycoplasma positive). Stable isotope labelling by amino acids in cell culture (SILAC) medium was prepared as follows: DMEM lacking lysine, arginine and methionine was custom prepared by AthenaES (Baltimore, MD, USA) and supplemented with 30 mg l⁻¹ methionine (Sigma-Aldrich; Oakville, ON, Canada), 10% (v/v) dialysed FBS (GIBCO-Invitrogen; Burlington, ON,Canada), 1 mM sodium pyruvate (Gibco-Invitrogen), 28 g ml⁻¹ gentamicin (Gibco-Invitrogen), and [¹³C₆,¹⁵N₂]-L-lysine, [¹³C₆,¹⁵N₄]-L-arginine (heavy isotopic form of amino acids; Heavy Media) from Sigma-Aldrich (Oakville, ON, Canada) at final concentrations of 42 and 146 mg l⁻¹ for arginine and lysine, respectively. For HCT-116, the concentration of arginine was increased to 84 mg l⁻¹. The cells were grown for at least 10 doublings in SILAC media to allow for complete incorporation of the isotopically labelled amino acids into the cells.