[35S]methionine-cysteine labeling and pulse-chase analysis.

For [³⁵S]methionine-cysteine labeling and pulse-chase analysis, the procedures described previously were followed with minor modifications (31). Briefly, A549 cells plated in a 60-mm dish at 80% confluence were starved for 1 h in methionine- and cysteine-free DMEM (Sigma) containing 2% dialyzed FBS. The cells were then metabolically labeled with 200 μCi of [³⁵S]methionine-cysteine (Express ³⁵S protein labeling mixture; PerkinElmer Life Sciences) for 15 min in methionine- and cysteine-free medium (pulse); unbound radioactive amino acids were washed and incubated with prewarmed complete medium (chase) in the presence of DMSO or 50 μM ibuprofen. The cells were then disrupted in ice-cold RIPA lysis buffer, and comparable amounts of cell extracts were immunoprecipitated with anti-AXL antibody. After washing 3 times, samples were subjected to SDS-PAGE, followed by transfer onto a nitrocellulose membrane. The labeled proteins were visualized by autoradiography.