Given the difference Semaxanib between our data and a previous report suggesting that laforin monomer is BEZ235 PI3K inhibitor inactive, we decided to determine a possible cause for this discrepancy. We found an interesting result when laforin was stored at 220uC in the absence of reducing agent. We purified monomeric laforin using Ni-NTA resin and size-exclusion chromatography by collecting peak B (Figure 1A), and then stored the purified protein at 220uC in the presence or absence of a reducing agent (10 mM DTT). These purified proteins were then reloaded onto an analytical sizeexclusion column (Superdex 200) and, in the case of proteins stored in the absence of DTT we observed multimeric species that separated as high molecular weight proteins (larger than 2,000 kDa) (Figure 4A; non-reducing peak). However, proteins stored in the presence of a reducing agent eluted as a single peak around 37 kDa (Figure 4A; reducing peak). Thus, monomeric laforin again remains as a monomer and does not convert into dimeric laforin similar to our findings in Figure 1B. The finding that storage of laforin in low levels of DTT is necessary prompted us to further examine the effect of reducing agents on laforin oligomerization and phosphatase activity. When we analyzed the non-reducing peak of laforin (Figure 4A) by gel electrophoresis under non-reducing conditions (no SDS and no DTT was present in the sample loading buffer and the samples were not heated), we observed the presence of laforin monomers, dimers, and multimers (Figure 4B, first lane). However, if we added increasing amounts of DTT we found that laforin oligomerization was reversed and at 100 mM DTT only monomeric laforin remained (Figure 4B). These results suggest that laforin oligomerization is very sensitive to oxidation, and that multiple species of laforin form under non-reducing conditions. These species may result from intermolecular disulphide bond formation among the nine cysteine residues present in laforin. Additionally, these results show that the amount of DTT commonly utilized in phosphatase assays (1-2 mM DTT) does not affect dimerization or multimerization. However, these low levels of DTT are necessary to keep the catalytic cysteine reduced.