Figure 5.
EPSIN1 Binds to VTI11.
(A) Protein extracts were prepared from VTI11:HA- and VTI12:HA-transformed protoplasts and mixed with GST alone or GST:EPSIN1. EPSIN1-bound proteins were precipitated from the mixture with glutathione–agarose beads and analyzed by protein gel blotting using anti-HA antibody.
(B) Coimmunoprecipitation of EPSIN1:GFP with VTI11:HA. Protein extracts from protoplasts cotransformed with VTI11:HA and EPSIN1:GFP were used for immunoprecipitation with anti-HA antibody. The immunoprecipitates were analyzed by protein gel blotting with anti-HA, anti-GFP, and anti-calreticulin antibodies. P, immunoprecipitate; S, supernatant; T, total protein extracts (5% of the input).
(C) For binding experiments, protein extracts from protoplasts transformed with VTI11:HA were mixed with GST alone, GST:ENTH, and GST:EPSIN1ΔN. Proteins were precipitated with glutathione-agarose beads and analyzed by protein gel blotting using anti-HA antibody. The amount of the input proteins is indicated.