These results demonstrate that the phosphatase activity of monomeric and dimeric laforin is both dependent on a reduced environment. In order to further probe the effect of reducing conditions on laforin dimerization, we analyzed the oligomeric status of laforin in mammalian HEK293 cells lysed in the presence and absence of reducing agent. When the cell extracts were prepared in the absence of DTT, clear monomeric, dimeric, and multimeric species were resolved by non-reducing gel electrophoresis. However, if cells were lysed in the presence of 10 mM DTT only monomeric laforin was detected. These results suggest that redox conditions may regulate laforin dimerization and that cellular oxidative stress may affect laforin oligomerization. It is important to note that these results strongly suggest that no laforin dimer is present with $10 mM DTT. The phosphatase assays performed in the presence of DTT employed 10 mM DTT in the assay buffer. Therefore, all of the laforin present should be in monomeric form and the monomeric laforin does possess phosphatase activity, supportive of our findings in Figure 2. Laforin dimerization does not affect glucan binding The dimer interface of laforin could involve the carbohydratebinding module, and if so dimerization could provide a mechanism to modulate glucan-binding. To test whether dimerization of laforin impacts its ability to bind glucans, we utilized a glucan-binding assay. In this assay, proteins are added to an amylopectin solution and the mixture undergoes ultracentrifugation. Proteins in the pellet and supernatant fractions are then assessed by Western analysis. Proteins with glucan-binding ability are retained in the pellet fraction and proteins lacking this ability are observed in the supernatant fraction. Immunoblotting of the pellet and supernatant fractions from the glucan-binding assay showed that both laforin monomer and dimer bind to amylopectin and are enriched in the pellet fraction. Therefore, dimerization of laforin does not inhibit its glucan-binding. Multiple groups have reported that glycogen inhibits laforin phosphatase activity. In agreement with these results, we observed a clear inhibition of monomeric laforin phosphatase activity in response to higher levels of glycogen in the reaction mixture. Similarly, we found that glycogen inhibits the phosphatase activity of dimeric laforin. As expected, glycogen did not affect the phosphatase activity of VHR, a dual specificity phosphatase that lacks a CBM. Therefore, glycogen inhibits the phosphatase activity of monomeric and dimeric laforin. As discussed in the introduction, laforin forms a functional complex by associating with malin and this complex is involved in ubiquitination and proteasomal degradation of multiple proteins involved in glycogen metabolism. The differences in the structure of monomeric and dimeric laforin could alter its ability to interact with malin that could change the scaffolding function of laforin. Therefore, we investigated the ability of malin to interact with monomeric or dimeric laforin by co-immunoprecipitation. We transfected HEK293 cells with FLAG-tagged malin, lysed the cells, immunoprecipitated FLAG-malin with anti-FLAG M2 agarose beads, and washed the beads multiple times. We then variety cell permeable low toxic fluorescence dna dyes commercially incubated the bound FLAG-malin with monomeric or dimeric laforin, again washed the beads, eluted bound proteins with NuPage sample buffer, and analyzed the proteins by Western analysis.