561862.fig.009a
(a)
561862.fig.009b
(b)
Figure 9: EGFR exposed to AECS/p-BQ cannot bind c-Cbl and is not ubiquitinated. Serum-starved A549 cells were either nontreated (NT) or exposed to 100 ng/mL EGF for 5 min (used as positive control), 2 μL/mL AECS or 200 ng/mL p-BQ for 1 hr. After treatment, cells were immediately lysed, and the EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with the indicated antibodies (a). Serum-starved A549 cells were either nontreated (NT) or exposed to 100 ng/mL EGF for 5 min or 2 hr, 2 μL/mL AECS or 200 ng/mL p-BQ for 1 hr. After treatment, cells were washed with PBS and further incubated in fresh serum-free medium for 2 hr at 37°C before lysis. The EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with antiphosphotyrosine (PY20) antibody (b).