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. 2010 Aug 9;190(3):307–315. doi: 10.1083/jcb.201005018

Figure 2.

Figure 2.

The recruitment of SHIP2 to CCPs is mediated by intersectin. (A–F) COS-7 cells transiently expressing GFP fusions of SHIP2 constructs as indicated together with either clathrin-mRFP or mCherry-actin. 5-Ptase, 5-phosphatase. (G) SDS-PAGE gel (Coomassie blue staining) of the material affinity purified from a rat brain extract by pull-downs with GST or GST–SHIP2-1022–1078 as indicated (ITSN-L and -S, long and short isoforms of intersectin). (H) Anti–intersectin 1 Western blotting of material pulled down from rat brain lysate with GST, GST–SHIP2-1022–1078 (WT) or GST–SHIP2-1022–1078 with an F to A mutation at position 1054 (FA). The GST proteins used for the pull-down are shown below the Western blot (Coomassie blue staining). The dashed line indicates separate membranes. (I) Western blotting with anti–intersectin 1 or anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies of COS-7 cells transfected with control siRNA (ctrl) or intersectin 1–specific siRNA (KD). (J) TIRFM image of COS-7 cell transfected with control siRNA (ctrl) or intersectin 1–specific siRNA (KD) transiently expressing GFP-SHIP2 and clathrin-mRFP. Bars, 5 µm.