Supplementary Figure 2: Characterization of recombinant PTEN protein in lipid and protein phosphatase assays.

(a) WT PTEN but not the CS or GE mutant dephosphorylates PIP3. The assay was performed as described in Methods. 40 μM PIP3 and 0.1 μM PTEN (WT), 10 μM PTEN (CS) and 10 μM PTEN (GE) proteins were used in the assay. WT: wild-type; CS: C124S mutant; GE: G129E mutant. (b) Coomassie blue gel showing purified recombinant PTEN proteins. YL: Y138L mutant. (c) WT-PTEN or GE but not YL or CS mutant can dephosphorylate IRS1. Immunoprecipitated IRS1 was incubated with recombinant PTEN (WT, YL GE or CS mutant) protein (0.6 μM) for 1 hr at 30 °C. (d) Comparing PTEN (WT or mutants) activity in lipid phosphatase assay. The assay was performed similar as in (a), with 40 μM PIP3 and PTEN (WT/YL/CS/GE) proteins of indicated concentrations. (e) Comparison of PTP1B and PTEN activity in the in vitro protein phosphatase assays. The assay was performed similar as in (c), with substrates (IGF1R and IRS1) and enzymes (PTP1B and PTEN) as indicated, and quantitated by densitometry.