4.10. SILAC Labeling and Protein Extraction

SILAC labeling was performed as described before [64,68]. Briefly, two cell populations of generated KM12-UPF3A clone were cultured separately in ’heavy medium (containing L-[¹³C₆, ¹⁵N₄] arginine (R10); L-[¹³C₆, ¹⁵N₂] lysine (K8)) or ‘light´ medium (containing L-[¹²C₆,¹⁴N₄] arginine (R0); L-[¹²C₆,¹⁴N₂] lysine (K0)) (Silantes, München, Germany) at 37 °C in a 5% CO₂ atmosphere for 8 days. To avoid arginine-to-proline conversion, the medium was additionally supplemented with L-proline (Sigma-Aldrich) to a final concentration of 200 µg/mL [69]. The saturated incorporation was confirmed as described below and shown at Supplementary Figure S4. ‘Heavy’ labeled cells were then treated for 24 h with doxycycline (500 ng/mL) to induce UPF3A expression, whereas control cell populations were exposed to dox-free medium. The experiment was performed in triplicate. For protein extraction, cells were suspended in a radioimmunoprecipitation assay buffer (RIPA), containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS supplemented with 1% DTT and fresh protease (cOmplete Mini; Roche) and phosphatase (PhosSTOP, Roche) inhibitors, and treated with benzonase (125 U; Merck Millipore, Burlington, MA, USA) on an orbital shaker (at 300 rpm) on ice for 1 h. After centrifugation at 13 000 rpm for 30 min at 4 °C protein concentration of the extracts was measured by using 2D Quant Kit reagents (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions.