Figure 7.

The laforin–malin complex promotes the aggregation of coexpressed AMPKβ subunits. (A–D) HEK293 cells were transfected with plasmid pCMVmyc-AMPKβ2 with (B and C) or without (A) plasmids pCMV-HA-laforin and pcDNA3-HA-malin (Laf+Mal). When indicated, cells were also transfected with plasmid pCMV-His6xUbiq K63R (D). The subcellular localization of AMPKβ2 subunit was carried out as described in Materials and Methods by using anti-AMPKβ total as primary and anti-rabbit Alexa-Fluor 488 as secondary antibodies. The same samples were treated with Topro3 to stain the nucleus and with anti-laforin or anti-ubiquitin as primary and anti-mouse Texas Red as secondary antibodies to determine the localization of laforin and ubiquitin conjugates. The three images were subjected to a merge analysis. (E) Quantification of cells expressing AMPKβ2 and showing either a punctuated distribution or large inclusion bodies. One hundred cells expressing AMPKβ2 from each of the above conditions were used to estimate the proportion of cells with or without inclusions. Bars indicate SD; statistical significance was considered at *p < 0.05 and **p < 0.01.