Figure 2

Immunoprecipitation and subsequent SELDI-TOF MS analysis of the target proteins. Extracts prepared from the A549 cells overexpressing 4E-BP1 were first incubated with anti-total-4E-BP1 antibody and then with protein G agarose. The bound proteins were eluted from the resins as described in Methods. Eluate from the untreated cells (a), eluate treated with λ-PPase for 1 hr at 30°C (b), and λ-PPase alone (c) were applied to NP20 chips and analyzed by SELDI-TOF MS. Lysate from the cells pre-treated with 1.5 μM ZSTK474 for 30 min was immunoprecipitated and processed for analysis by SELDI-TOF MS (d). The left-right arrow indicates a mass difference of 80 Da.