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Fig. 3. SNX9, SNX18 and SNX30 interact and colocalize with dynamin 2. (A) GST or GST-fusion proteins with SH3-domains from SNX9, SNX18 or SNX30 were incubated with (+) or without (–) cytosol from HeLa cells. After washings, bound protein was analyzed by SDS-PAGE and the gel was stained with Coomassie. The identity of dynamin 2 (Dyn2) was verified with immunoblotting. (B) The PRD of dynamin 2, containing wild-type (wt) or mutated (mut1-4) sequences, was purified and used in pull-downs with GST or GST-fusion proteins containing SH3-domains from SNX9, SNX18, SNX30 or amphiphysin 2 (Amph2), as described in the Materials and Methods. Bound protein, together with total protein (Input), was analyzed by SDS-PAGE and the gels were stained with Coomassie. The sequence of the C-terminal part of the PRD with indicated type I and type II SH3-binding motifs is shown below. Modified amino acids in mutated PRDs are highlighted. (C) Epifluorescent micrograph of HeLa cells expressing myc-tagged SNX9 (a) or myc-tagged SNX18 (b) and counterstained for dynamin 2 after wash-out of cytosolic components as described in the Materials and Methods. Magnifications of the boxed areas together with illustrations of the specific structures labeled with anti-myc and anti-dynamin-2 are shown to the right. Scale bar: 10 µm.
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