Fig. 3

Interaction of PDZK1 with SMCT1 and SMCT2. a Full-length PDZK1 PCR product was in vitro-translated in the presence of Transcend Biotinylated Lysine tRNA (Promega). The in vitro translation products were incubated with GST alone (lane 1), GST-SMCT1-CTwt (lane 2), GST-SMCT1-CTd3 (lane 3), GST-SMCT1-CT-T608A (lane 4), or GST-SMCT1-CT-L610A (lane 5) in a Glutathione Sepharose 4B affinity resin (GE Healthcare). The pull-down products were analyzed by SDS-PAGE. The input corresponds to the crude in vitro translation reaction. Positions of molecular mass standards are indicated on the left. GST fused to SMCT1 C-terminal wild type can coprecipitate PDZK1, confirming the specificity found in the yeast two-hybrid system. b Full-length PDZK1 PCR product was in vitro-translated in the presence of Transcend Biotinylated Lysine tRNA (Promega). The in vitro translation products were incubated with GST alone (lane 1), GST-SMCT2-CTwt (lane 2), GST-SMCT2-CTd3 (lane 3), GST-SMCT2-CT-T616A (lane 4), or GST-SMCT2-CT-F618A (lane 5) in a Glutathione Sepharose 4B affinity resin (GE Healthcare). The pull-down products were analyzed by SDS-PAGE. The input corresponds to the crude in vitro translation reaction. Positions of molecular mass standards are indicated on the left. GST fused to SMCT2 C-terminal wild type can coprecipitate PDZK1, confirming the specificity found in the yeast two-hybrid system