Fig. 5. The N-terminal region of RIN3 containing PRDs specifically interacts with the SH3 domain of amphiphysin II. (A) The full-length (FL) or SH3 domain (SH3) of GST-fused amphiphysin II (GST-amph II) or GST alone was incubated with Flag-RIN3 purified from baculovirus-infected Sf9 cells and glutathione resin. Proteins bound to the resin were separated by SDS-PAGE and immunoblotted (IB) with anti-Flag (top) and anti-GST (bottom) antibodies. (B) Lysates were prepared from HeLa cells expressing RIN3 and the full length (FL) or SH3-domain-deleted form (SH3) of Flag-tagged amphiphysin II (Flag-amph II) and incubated with glutathione resin. Proteins bound to the resin were separated by SDS-PAGE and immunoblotted with anti-RIN3 (top) and anti-Flag (bottom) antibodies. (C) Lysates were prepared from HeLa cells expressing the full length (FL), N-terminal (N) or C-terminal (C) form of Flag-tagged RIN3 (Flag-RIN3) and incubated with GST-fused amphiphysin II (GST-amph II) and glutathione resin. Proteins bound to the resin (GST-pull down) and the total lysate were separated by SDS-PAGE and immunoblotted with anti-Flag (top) and anti-GST (bottom) antibodies. (D) Flag-RIN1 and RIN3 were purified from baculovirus-infected Sf9 cells, and GST pull-down assay was performed as described in (A). Proteins bound to the resin (GST pull-down) and the total lysate were separated by SDS-PAGE and immunoblotted with anti-Flag (top) and anti-GST (bottom) antibodies.

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