Skip to main content
. 2010 Feb 19;285(16):12279–12288. doi: 10.1074/jbc.M109.086405

FIGURE 2.

FIGURE 2.

The C2 domain of Nedd4 interacts with its HECT domain in the absence of calcium. GST-C2 domain pulldown assay for binding of the C2 domain to purified T7-tagged HECT domain of Nedd4-2a (A), or HA-tagged Nedd4-2bΔN (B). The C2 domain of Nedd4-2 was subcloned into a GST fusion protein expression vector, expressed in E. coli, and purified by affinity purification with glutathione-conjugated agarose beads. The beads with GST (control) or GST-Nedd4-2b C2 domain fusion protein were incubated with purified T7-tagged HECT domain of Nedd4-2a in indicated calcium concentrations, or with HA-Nedd4-2bΔN- or HA-Nedd4-2bWW-expressed HEK293 cell lysates in the presence of 5 mm CaCl2 or 1 mm EGTA. The GST-C2 domain-precipitated T7-Nedd4-2a-HECT, HA-Nedd4-2bΔN, or HA-Nedd4-2bWW was detected by immunoblotting with anti-T7 antibody or anti-HA antibody. The GST and GST fusion proteins used for the pulldown assay are shown by Coomassie Blue staining. Lane 1 in A and B shows one-tenth the total input of the indicated proteins in the pulldown assay.