Figure 7.

TRIM32 ubiquitinates another malin substrate such as AMPK subunits. A) HEK293 cells were co-transfected as in part A of Figure 6 using myc-AMPK subunits α2, β2 or γ1 as a substrate, and either Flag-TRIM32 or empty vector (Flag). Samples were processed and analyzed as in Figure 6. Only in the case of AMPKα2 (left panel) and AMPKβ2 (middle panel), Flag-TRIM32 produced poly-ubiquitinated forms. B) Chain topology of ubiquitinated AMPK subunits. HEK293 cells were co-transfected with myc-AMPKα2 (left panel) or β2 subunit (right panel), 6xHis-tagged ubiquitin constructs (WT, K48R or K63R), and either empty vector (Flag) or Flag-TRIM32. Cell extracts were analyzed as in Figure 6 using anti-myc antibodies.