Fig. 6

Multimolecular complex formation including SMCT2-PDZK1-URAT1. GST-fused SMCT2-CTwt was incubated together with in vitro translated full length PDZK1 and MBP-fused URAT1-CTwt in a Glutathione Sephrarose affinity column and were eluted and analyzed by SDS PAGE. The full length PDZK1 PCR product was in vitro translated in the presence of Transcend biotinylated lysine tRNA (Promega). The in vitro translated products were incubated with MBP-URAT1-CTwt (lane 1), GST alone (lane 2), positive control: GST-URAT1-CTwt alone (lane 5), GST-SMCT2-CTwt (lane 6), GST-SMCT2-CTwt and MBP-URAT1-CTwt (lane 7), negative controls were GST alone and MBP-URAT1-CTwt without PDZK1 (lane 3) and GST-SMCT2-CTwt and MBP-URAT1-CTwt without PDZKI (lane 4). GST-fused SMCT2-CTwt can coprecipitate PDZK1 along with MBP-fused URAT1-CTwt. MBP-URAT1-CTwt could also be precipitated with GST-SMCT2-CTwt in the absence of PDZK1, confirming that PDZK1 interacts with SMCT2-CT and may not be necessary for the interaction between URAT1 and SMCT1. The input corresponds to the crude in vitro translation reaction and MBP-URAT1-CT lysate. The positions of molecular mass standards are shown on the left