Therefore, all of the laforin present should be in monomeric form and the monomeric laforin does possess CPI-613 Dehydrogenase inhibitor phosphatase activity, supportive of our findings in Figure 2. The dimer interface of laforin could involve the carbohydratebinding module (CBM), 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 showed that both laforin monomer and dimer bind to amylopectin and are enriched in the pellet fraction (Figure 5A). 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 (Figure 5B). Similarly, we found that glycogen inhibits the phosphatase activity of dimeric laforin (Figure 5B). As expected, glycogen did not affect the phosphatase activity of VHR (Figure 5C), 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 NVP-BKM120 PI3K inhibitor 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.