Phosphorylation of Bim_EL at serine 65 promotes Bim_EL-mediated apoptosis. A, Cerebellar granule neurons were transfected with control vector (V), Bim_EL, or Bim_ELS55/65A (55/65A) and β-galactosidase (left panel) or with control vector (V) or JNK DN together with Bim_EL and β-galactosidase (right panel). One day after transfection, cultures were switched from full medium to basal medium in the absence of survival factors. Cells were fixed after 8 hr and subjected to indirect immunofluorescence. Percentage of apoptosis is represented as mean ± SE. The expression of Bim_ELS55/65A induced significantly less apoptosis than Bim_EL (n = 3; ANOVA; p < 0.0005). Expression of JNK DN significantly reduced Bim_EL-induced apoptosis (n = 3; ANOVA; p < 0.005). B, Cerebellar granule neurons were transfected with plasmids encoding nonspliceable cDNAs of Bim_EL, Bim_ELS55A (55A), Bim_ELS65A (65A), Bim_ELS65D (65D), or control vector (V) and β-galactosidase. Cultures were treated as described above and analyzed by indirect immunofluorescence. The expression of Bim_ELS65A induced significantly less apoptosis than Bim_EL and Bim_ELS65D (mean ± SE; n = 4; ANOVA; p < 0.01 and p < 0.0005). The expression of Bim_ELS55A did not reduce the apoptotic effect compared with wild-type Bim_EL (mean ± SE; n = 6; ANOVA; p = 0.4). C, Cerebellar granule neurons were transfected with plasmids encoding nonspliceable cDNAs of wild-type Bim_EL, Bim_ELS65D (65D), or control vector (V) and β-galactosidase. One day after transfection, cultures were switched from full medium to basal medium in the presence (Ins) or absence (-) of insulin (10 μg/ml) to activate the IGF-I receptor. Cells were fixed after 8 hr and subjected to indirect immunofluorescence. Activation of the IGF-I receptor reduced Bim-mediated apoptosis (mean ± SE; n = 4; ANOVA; p < 0.0001) but had little effect on Bim_ELS65D-induced apoptosis (p > 0.04).