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. 2014 Apr 28;205(2):197–215. doi: 10.1083/jcb.201307088

Figure 4.

Figure 4.

Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp IΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.