Inhibition of Methionine Aminopeptidases by Bengamide A Changes c-Src N-terminal Myristoylation Status.
Immunoprecipitation of C-terminal myc-tagged c-Src protein from HEK293 cell lysate was aliquoted either for western blotting by anti-Myc antibody (A), or for [35S]-methionine scintillation counting (B) after in vitro processing by both MetAP1 and MetAP2. Bengamide A (10 nM) was added for the last 18 hours. Immunoprecipitation from [3H]-myristic acid-labeled HEK293 cell lysate were aliquoted either for western blot (C, E) or for [3H] scintillation counting as an indication of N-myristoylation (D, F). CHX, cycloheximide (4 μg/mL); Beng A, bengamide A (100 nM) and 5-FU, 5-fluorouracil (100 μM), IV-43 (10 μM), TNP-470 (100 nM) and the combination of IV-43 (10 μM) and TNP-470 (100 nM), were added to cell media for the last 6 hours.