N n diisopropylcarbodiimide dic

HEK 293 cells were purchased from ATCC while the stably transfected HEK 293-hUT cell line was generated in our laboratory (23 ). Cell culture reagents were obtained from Invitrogen while antibodies used to evaluate ERK_1/2 phosphorylation, i.e., a rabbit polyclonal antibody against phospho-p44/42 MAPK and an anti-total MAP kinase antibody, were purchased from Cell Signaling Technology. α-Actin antibody was obtained from Abcam. The IP-One ELISA assay kit from CisBio Bioassays. The fluorenylmethyloxycarbamate- (Fmoc-) protected amino-acids, Rink amide AM resin (with Nle), N,N′-diisopropylcarbodiimide (DIC), and ethyl cyanohydroxyiminoacetate (Oxyma) were purchased from Chem-Impex. Trifluoroacetic acid (TFA), methanol (MeOH), acetonitrile (ACN), diethyl ether, N,N-dimethylformamide (DMF), piperidine, dichloromethane (DCM), and cell mask were obtained from Fisher Scientific. YM254890 was purchased from Cedarlane. All other chemicals, including 99.9% ²H₂O, were from Sigma-Aldrich. 98% DPC-d₃₈ was obtained from Cambridge Isotope Laboratories, Inc, and [(2,2,3,3-tetradeuterio)-3-(trimethylsilanyl)]propionic acid (TSP) from MSD Isotopes.

Reagents and solvents were purchased from commercial vendors and used without further purification. Rink amide MBHA resin, 2‐chlorotrityl resin, and Fmoc‐protected amino acids were purchased from Merck Millipore (Billerica, MA, USA). Trifluoroacetic acid (TFA) and N,N‐dimethylformamide (DMF, >99.9%, peptide synthesis grade) were purchased from Acros Organics (Fair Lawn, NJ, USA). Acetonitrile (ACN, HPLC grade), propidium iodide (PI), and FM4‐64 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). N,N’‐Diisopropylcarbodiimide (DIC) was purchased from Chem‐Impex (Wood Dale, IL, USA). 1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE), 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC), 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐(1’‐rac‐glycerol) (POPG), 2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(10‐rac‐glycerol) (DMPG), and 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol)‐2000] (DMPE‐PEG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Mueller‐Hinton broth 2 (MHB2), bovine serum albumin (BSA), thioflavin T (Th T), thioflavin S (Th S), acetic acid, poly‐L‐lysine solution, and human liver S9 were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Other chemical reagents and solvents were purchased from TCI (Tokyo, Japan) and Alfa Aesar (Haverhill, MA, USA).