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. 2004 Oct 14;101(43):15313–15317. doi: 10.1073/pnas.0406837101

Fig. 4.

Fig. 4.

Phosphorylation of BIM by constitutive active MEK counters IL-3 withdrawal death. (A) FL5.12 BCL-2/BIM WT or BCL-2/BIM S55AS65AS100A cells were transfected with either GFP (control) or GFP-constitutively active MEK1 (CA-MEK) plasmids, respectively. Total cell extracts prepared at 24 h after IL-3 deprivation were subjected to Western blots with anti-phospho-S65 BIM and anti-HA (for total BIM and CA-MEK, respectively). (B) Transfected cells from above were deprived of IL-3 for 24 h, then stained with PI to identify nonviable cells, and analyzed by FACS, gating on GFP-positive transfected cells. Percentage protection relative to controls was calculated as 100 × [1 - (percentage apoptosis of cells transfected with CA-MEK plasmid/percentage apoptosis of cells transfected with control plasmid)]. Results are the mean ± SD of two independent experiments.