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Lack of correlation between SH3 binding, stimulation and assembly of dynamin.

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posted on 10.12.2015, 10:38 authored by Sai Krishnan, Michael Collett, Phillip J. Robinson

(A) The relative binding of dynamin to individual GST-SH3 domains (~12 μg) was compared by performing pull-down analysis using purified sheep dynamin I (~3 μg) in the presence of low or high salt. The pull-down was resolved on 10% SDS-acrylamide gels and a Coomassie Blue stained gel is shown. The image are representative of n = 3 independent experiments. (B) The effect of purified recombinant GST-SH3 domains (40 μg/ml) on stimulation of purified sheep brain dynamin I (100 nM / 10 μg/ml) GTPase activity using ELIPA in the presence of low (30 mM NaCl) or high (150 mM NaCl) salt. All data is mean ± S.E.M. and n ≥ 3. The inset shows a zoom of the low and high salt values for GST-SNX9 SH3. A two-tailed Student t-test showed significance (*, p < 0.05). (C) Relative ability of 13 GST-SH3 domains to oligomerise dynamin using a sedimentation assay. GST-SH3 domain stimulated dynamin I assembly obtained from densitometric analysis of the dynamin pellet (see S2 Fig). The data is representative of n = 3 independent experiments performed in duplicate and is expressed as fold change relative to dynamin basal (no SH3) control.