Figure 4. ERK1/2-dependent Bim_EL turnover proceeds normally in the presence or absence of Cbl.

(A) Cycling c-Cbl-/- and c-Cbl-/- HA-c-Cbl reconstituted iMEFs were serum starved overnight to elevate levels of Bim_EL. Cells were then pre-treated with emetine for 30 minutes before being stimulated with FBS and harvested after 2, 4 or 8 hours. Whole cell extracts were prepared, normalised for protein content and boiled directly in sample buffer. Lysates were fractionated by SDS-PAGE and immunoblotted with antobodies to Bim, Cbl, P-ERK1/2 and ERK1/2, in which ERK1/2 served as a loading control. The immunoblots shown are from a single experiment representative of three giving identical results. (B) The amount of Bim_EL in the lysates relative to the ERK1/2 loading control was quantified by densitometric analysis. Data from three independent experiments was combined and plotted, confirming that the turnover of Bim_EL in the c-Cbl-/- MEFs did not differ significantly from that in the HA-c-Cbl addback cell line. (C) Cycling c-Cbl-/- iMEFs were serum starved overnight to elevate levels of Bim_EL. Cells were then pre-treated with emetine and MG132 for 30 minutes before being treated with FBS and harvested at set time intervals. (D) Cycling c-Cbl-/- were serum starved overnight to elevate levels of Bim_EL. Cells were then pre-treated with emetine and AZD6244 for 30 minutes before harvested at set time intervals. In both (C) & (D) whole cell extracts were prepared, normalised for protein content and boiled directly in sample buffer. Lysates were fractionated by SDS-PAGE and immunoblotted with antibodies to Bim, P-ERK1/2 and ERK1/2, the latter serving as a loading control. Results are taken from a single representative experiment; similar results were obtained in three experiments.