Figure 3.

Phosphorylation at Ser183 by PIM1 increases ABI2 stability. (a) 293T cells were transfected with GFP-ABI2 WT, S183A, or S183D prior to treatment with cycloheximide (CHX, 10 μM), and GFP levels were assessed by Western blotting. Densitometry was used to determine the rate of protein decay. (b and c) PC3-LN4 cells transfected with control vector or human PIM1 under normoxia (c) PC3-LN4 and PIM1-KO cells were treated with CHX for the indicated times at 1% O₂, and ABI2 levels were assessed by Western blotting. (d) PC3-dox inducible PIM1 cells were transfected with GFP-tagged WT-ABI2 or GFP-tagged S183A-ABI2 and treated with DMSO (−) or dox (+) (100 ng/ml) for 24 h. Protein levels were measured by Western blotting. (e) DU145 cells were incubated in normoxia or hypoxia (1.0% O₂) for 4 h prior to treatment. DMSO or PIM447 was added 30 min prior to addition of CHX, and cells were lysed at the indicated time points. (f) DU145 cells were transfected with GFP-ABI2 WT, S183A, and S183D under normoxic and hypoxic conditions in the presence or absence of PIM447. *P < 0.05, two-sided Student’s t test, n = 3 for all experiments, and error bars = SEM. Source data are available for this figure: SourceData F3.