Figure 3. UBA domain of AMPK-related kinases do not bind tetra-ubiquitin or polyubiquitin.

(A) GST-fusion proteins of the indicated UBA domains of AMPK subfamily kinases or the tandem UBA domains of Rhp23 bound to glutathione–Sepharose were incubated with either tetra-ubiquitin or K⁴⁸-linked polyubiquitin. The beads were washed and the associated protein was eluted and subjected to PAGE. The gels were either stained with Coomassie Blue (lower panel) to visualize levels of GST-fusion proteins or immunoblotted with anti-ubiquitin antibody to detect associated ubiquitin forms (upper panels). (B) As above, except that full-length GST-fusion proteins of AMPK subfamily kinases were employed rather than isolated UBA domains. The full-length SAKS1 ubiquitin-binding UBA domain was employed as a control [23]. The level of GST-fusion proteins associated with the glutathione–Sepharose was assessed by immunoblot analysis employing a anti-GST antibody (lower panel). The results are representative of two or three separate experiments.