Figure 2. Mutation of all four tyrosine residues does not affect the ability of Bim_EL to undergo ERK1/2-dependent phosphorylation and degradation.

(A) HR1 cells were transfected with HA-Bim_EL, HA-Bim_ELΔTyr or empty vector (EV) for 16 hours. Cells were then serum starved for 30 minutes in the presence of MG132 and subsequently treated with pervanadate for 10 minutes (see materials and methods). Whole cell extracts were then prepared, normalised for protein content, and boiled directly (input) or used for immunoprecipitaion of HA-Bim_EL. Input and IP samples were then immunoblotted with antibodies to anti-phosphotyrosine, Bim, HA (Bim), P-ERK1/2 and ERK1/2. Input lysates were included with the IP samples to act as a positive control for the anti-phosphotyrosine immunoblot. Note: the arrow in (A) indicates the position at which Bim resolves and would appear if it reacted with the anti-P-Tyr antibody. (B) HR1 cells were transfected with HA-Bim_EL or HA-Bim_ELΔTyr for 16 hours, then switched to serum free medium in the presence of emetine for a 30 minute pre-treatments. Cells were then stimulated with 100nM 4-HT for 2, 4 or 8 hours after which whole cell lysates were prepared for immunoblot analysis using antibodies to HA (Bim), P-ERK1/2 and ERK1/2.