GST Pull-Down Assay Protocol

His-E1, GST-WWP-1 (WT and C762A mutant) and UBC-18 recombinant proteins haw been previously described^{13 (link)}. The WW domain-containing region of WWP-1 (Amino acids 215–412) was cloned by PCR and subcloned into the pGEX6Pl bacterial expression vector to generate GST-WW. GST purifications were performed according to the manufacturer’s instructions, kit-1 andpha-4 cDNAs were subcloned into the pCRII vector (Invitrogen) and used in the TnT Coupled Transcriptionn/Translation Systems (Promega) to produce ³⁵S-methionine-labellcd protein. For pull-down experiments, 5 or 10 μl of ³⁵S-labclled lysates were incubated with 3.3 μg of GST or GST fusion protein bound to glutathione beads in binding buffer (50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 1% NP40, 10 mM MgCl, and 1 mM dithiothreitol) for 2 h at 4°C. Beads were washed four times with binding buffer and analysed by SDS-Polyacrylamide gel electrophoresis. ³⁵S-labelled proteins were identified by autoradiography.