Identification of major binding
partners of syndapin I as dynamin, synaptojanin, synapsin I, and
N-WASP. Soluble proteins (S3) from rat brains after homogenization in
buffer A were affinity purified onto either wild-type or mutant
GST-SdpI-SH3 immobilized on glutathione-Sepharose. Material
specifically bound to the beads was eluted with glutathione-containing
buffer and analyzed on 4–15% SDS-PAGE. (A) Coomassie protein staining
revealed a protein doublet at 100 kDa and a minor band at 145 kDa
specifically coprecipitated with wild-type SdpI-SH3 but not with
SdpI-SH3m harboring the P434L point mutation. (B) Precipitated material
was analyzed by overlay with GST-SdpI and by Western blot analyses.
Antibodies against dynamin, synaptojanin, and synapsin I demonstrate
that the affinity-purified bands of 100, 145, and 80 and 75 kDa
correspond to dynamin, synaptojanin, and synapsin Ia and Ib,
respectively. N-WASP–specific antibodies revealed that syndapin I
coprecipitated N-WASP because of an SH3 domain-dependent interaction.