4.4. In Vitro Ubiquitination Assays

MuRF1 and MuRF2 full-length human cDNAs [45] were inserted into pETM-44 and expressed in Escherichia coli as hexahistidine maltose-binding protein (MBP) fusions. Shortly before setting up the ubiquitination reactions, the MBP tag was removed from MuRF1 and MuRF2 by proteolytic cleavage with precision protease (obtained from the European Molecular Biology Laboratory protein expression core facility). Human PDK2 full-length cDNA [46] was inserted into pETM-11 and expressed in E.coli as a hexahistidine-tagged fusion protein. Ubiquitination reactions were started by the addition of 75-nM E1, 1-µM E2 (UbcH5c), 150-µM ubiquitin, 4-µM ATP in 20-mM Tris/Cl, pH 7.5, 20-mM KCl, 5-mM MgCl2, 1-mM DTT, and 1-mM PMSF (phenylmethylsulfonylfluorid). E1, E2, and ubiquitin were purchased from BostonBiochem (Cambridge, MA, USA). Where indicated, PDK2 (2 µM), MuRF1 (1µM), or MuRF2 (1µM) were also added. Reactions were incubated for 1 h at 37 °C and then stopped by the addition of SDS-loading buffer. Samples were then analyzed by SDS-PAGE and blotted onto nitrocellulose. Specific products were detected with a rabbit polyclonal antiserum against PDK2 (Abcam, Cambridge, UK, product 68164).