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Fig. 1. Biochemical characterization of PTEN chimeras. (A) Extracts from Jurkat cells transfected with mock, N- or C-terminus PTEN chimeras or a bicistronic plasmid encoding PTEN or PTEN-C/S and GFP, were assayed for in vitro lipid phosphatase activity using PtdIns(3,4,5)P₃ as a substrate in a Malachite Green-based assay. The phosphate released (measured in pmoles) by the mock cell lysate was considered the reference unit. A western blot showing the corresponding cell extract PTEN levels is shown at the right. Data are representative of three separate experiments. (B) Extracts from unstimulated and CXCL12 (100 nM)-stimulated mock, PTEN-GFP- or PTEN-C/S-GFP-Jurkat cells were analyzed sequentially with anti-phosphoAKT and AKT Ab, and with anti-PTEN as a protein expression control. After densitometry, AKT phosphorylation was determined as the phospho-AKT:AKT ratio (x10) (n=3). (C) The AKT phosphorylation index was also determined using extracts from unstimulated and CXCL12-stimulated mock, GFP-PTEN or PTEN-GFP-Jurkat cells as above (n=2).

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