Identification of BIMEL phosphorylation sites in CLL and Ramos cells. (A) Ramos and CLL 332 cells were treated with anti-IgM for 30 minutes. Expression of S69-phosphorylated BIM and β-actin was analyzed by immunoblotting. (B) Ramos cells were transfected with wild-type rat HA-tagged BIMEL or S65 → A mutant rat HA-BIMEL expression plasmids, or were mock transfected as a control. After 24 hours, cells were treated with anti-IgM in the presence or absence of U0126 and PD0325901. Expression of HA-tagged BIM, phospho-ERK1/2, and β-actin was analyzed 30 minutes after IgM stimulation by immunoblotting. Open arrowheads indicate phosphorylated BIM. Data are representative of 3 independent experiments. (C) Ramos cells were transfected with wild-type rat HA-BIMEL or mutant rat HA-BIMEL expression plasmids and 24 hours later treated with anti-IgM. After 30 minutes, expression of HA-tagged BIM and β-actin was analyzed by immunoblotting. Open arrowheads indicate phosphorylated BIM. Data are representative of 3 independent experiments. pAII indicates rat HA-BIMEL with combined mutations at amino-acid residues 55, 65, 73, 100, 112, and 114.
