Ubch5b ube2d2

HA-Ub (Boston Biochem), Ubiquitin Activating Enzyme (UBE1) (Boston Biochem) and UbcH5b/UBE2D2 (Boston Biochem) were obtained. Recombinant WT Nedd4-2 was obtained from Abcam. When HA-tagged WT or mutant Nedd4-2s were produced in HEK cells, 250 μg of total protein lysates were subjected to immunoprecipitation with anti-HA antibody to partially purify HA-tagged Nedd4-2s. Recombinant GluA1 (Origene) was used as substrate for in vitro ubiquitination with recombinant Nedd4-2 (Fig 7A) or Nedd4-2s obtained from transfected HEK cells (Figs 6B and 7C) following a protocol previously described [63 (link)]. Recombinant 14-3-3ε was obtained from Origene.

In vitro ubiquitination assays were performed as described previously [37] (link) with slight modifications. In brief, the reaction mixture contained 100 nM recombinant human E1 (His-UBE1, Cat No. E-304, Boston Biochem, Cambridge, MA), 1.4 μM recombinant human E2 (UbcH5b/UBE2D2, Cat No. E2-622, Boston Biochem), and 30 μM recombinant human HA-ubiquitin (Cat No. U-110, Boston Biochem) in ubiquitination buffer [25 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 2.5 mM ATP, and 4 mM MgCl₂]. The recombinant human His-RNF168/His-RNF168(CC16/19SS) and GST-RNF126/GST-RNF126 (CC229/232AA) proteins expressed in E. coli were made in the lab and included to a final volume of 50 μl with a final concentration of 1 μM. The reaction mixtures were incubated for 60 min at 30 °C and terminated by adding 5× SDS sample buffer.