Overlay analysis of rat brain cytosol with various
GST-syndapin I constructs. Rat brain cytosol (20 μg per lane) was
separated on 4–15% SDS-PAGE gels, transferred to nitrocellulose
membranes, and either overlaid with 1 μM GST fusion proteins of
syndapin I (wild-type and P434L mutant of full-length syndapin I and
SH3 domain only and SdpI-N, lacking the SH3 domain) or incubated with
antibodies against synapsin I, dynamin (2072), and synaptojanin.
Binding of GST fusion proteins was detected with affinity-purified
anti-GST antibodies. Full-length syndapin I fusion protein (GST-SdpI)
reveals major bands at 170 kDa, 145 kDa (comigrating with
synaptojanin), 120 kDa, 100 kDa (comigrating with dynamin), 80 and 75
kDa (doublet comigrating with synapsin Ia and Ib), 65 kDa, and 55 kDa,
whereas the SH3 domain showed a high preference for the 100-kDa band
corresponding to dynamin (GST-SdpI-SH3).