TBC1D12 interacts with Rab11 through its middle region.

<p>(A) TBC1D12 interacts with a constitutively active form that mimics the GTP-bound form of Rab11. Total lysates of COS-7 cells expressing T7-TBC1D12 were incubated with glutathione-Sepharose beads coupled with GST alone, GST-Rab11A-Q70L (indicated by QL), or GST-Rab11A-S25N (indicated by SN). A 2% volume of the reaction mixture (input) and proteins bound to the glutathione-Sepharose beads were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (top and middle panels), and the GST-fusion proteins were stained with amido black (bottom panel). The positions of the molecular mass markers (in kDa) are shown on the left. (B) Schematic representation of three deletion mutants of TBC1D12 (TBC1D12-N, -M, and -TBC). TBC domains and coiled-coil (CC) domains are shown as blue boxes and green boxes, respectively. (C) Total lysates of COS-7 cells expressing T7-TBC1D12 (indicated by FL, full-length), T7-TBC1D12-N, T7-TBC1D12-M, or T7-TBC1D12-TBC were incubated with glutathione-Sepharose beads coupled with GST alone (indicated by G) or GST-Rab11A-Q70L (indicated by 11). A 2% volume of the reaction mixture (input) and proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (top three panels). The third panel corresponds to the longer exposure of the second panel. The GST-fusion proteins were stained with amido black (bottom panel). The asterisk indicates non-specific bands, and the positions of the molecular mass markers (in kDa) are shown on the left.