Methionine met

Hepes, methionine (Met), phenylmethanesulfonyl fluoride, and 5-formyltetrahydrofolate (folinic acid (FnA)) were obtained from Sigma-Aldrich (St Louis, MO, USA). l-glutamine was purchased from Biochrom-AG (Berlin, Germany). Newborn calf bovine serum and endothelial cell growth factor were from Roche (Mannheim, Germany) and collagenase, M199 basal culture medium (with Earle’s balanced salt solution and Hepes), and Hank’s balanced salt solution were from Promocell (Heidelberg, Germany).

eIF2α-P-deficient MEFs were seeded at 5 × 10⁴ cells/well in 24-well plates. Cells were grown overnight and treated as described above. Prior to the end of each treatment, [³⁵S]-Met/Cys was added (30 μCi/mL EXPRE³⁵S Protein Labeling Mix, PerkinElmer) for 30 minutes. Next, cells were washed twice with cold phosphate-buffer saline (PBS) and total proteins were precipitated in 5% trichloroacetic acid (TCA) with 1 mM Methionine (Met) (Sigma-Aldrich) for 15 minutes on ice. The precipitation step was repeated overnight at 4°C. After careful removal of TCA-Met, 200 μL of 1 N NaOH and 0.5% sodium deoxycholate were added for 30 minutes. To determine the incorporation of [³⁵S]-Met/Cys into total cellular proteins, liquid scintillation counting, and DC Protein Assay (Bio-Rad) were used to quantify radioactivity and protein concentration, respectively.

All chemical compounds employed in this paper were of analytical grade. Silver nitrate ( ${\mathrm{AgNO}}_3$ ) and trisodium citrate were purchased from Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany), respectively. The aqueous solutions were prepared in Milli-Q water (Milli-Q® Direct Water Purification System, Darmstadt, Germany). ${\mathrm{MgF}}_2$ polished glasses (Crystran Ltd., Poole, UK), having a diameter of 20 mm, were used as port probes for the creation of the solid SERS substrates. The analytes and the pharmaceutical compounds used for SERS measurements (methylene blue MB, doxorubicin DOX, atenolol ATE, metoprolol MET, cysteine Cys and methionine Met) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture products: Roswell Park Memorial Institute (RPMI 1640) cell culture medium, Fetal Bovine Serum (FBS), Glutamine, Penicillin/Streptomycin and Phosphate Buffered Saline solution (PBS 1X) were purchased from Gibco (Grand Island, NY, USA).