JNK phosphorylates Bim_EL at the distinct site of serine 65 in vitro. A, Recombinant GST-Bim_EL (WT) and recombinant Bim_EL phosphorylation mutants GST-Bim_ELS55A (55A), GST-Bim_ELS65A (65A), and GST-Bim_ELT112A (112A) were incubated with no kinase or recombinant JNK1α1 and subjected to an in vitro kinase assay using [γ-³²P]ATP. Phosphorylated substrates were separated by SDS-PAGE, stained with Coomassie blue (bottom panel), and analyzed by autoradiography (top panel). Degradation products of bacterially expressed GST-Bim_EL are indicated with an asterisk. B, Recombinant GST, GST-Bim_EL (WT), and GST-Bim_ELS65A (65A) were subjected to an in vitro kinase assay with recombinant JNK1α1. Phosphorylated substrates were separated by SDS-PAGE and immunoblotted with the phospho65-Bim, Bim, or GST antibody. Degradation products of bacterially expressed proteins are marked with an asterisk.