spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 1


Fig. 1. Myosin VIIa binds to shroom2. (A,B) Domain structures of (A) myosin VIIa and (B) shroom2 (mShrm2). The domain composition and position of the yeast two-hybrid bait (myosin-VIIa–SH3-MyTH4-FERM) and of the prey clone (mShrm2MBR) are indicated. (B) Shroom2 consists of an N-terminal PDZ domain, an SPR, an MBR, and the ASD1 and ASD2 domains. (C) In transfected HeLa cells expressing GFP-tagged shroom2 MBR (GFP-mShrm2MBR; prey) alone, the protein exhibits a punctate cytoplasmic staining and is also particularly abundant in cytoplasmic extensions (arrowheads). (D) In co-transfected HeLa cells expressing both the prey GFP-mShrm2MBR (left panel) and Myosin VIIa tail (right panel), the two proteins colocalize in punctate structures (arrows) in the cytoplasm, arrowhead indicates cytoplasmic extension. (E) In transfected HeLa cells expressing GFP-tagged mShrm2MBR{Delta}C (GFP-mShrm2MBR{Delta}C, aa 350-563) alone, the fusion protein is diffusely distributed throughout the cytoplasm. (F) In co-transfected HeLa cells expressing both GFP-mShrm2MBR{Delta}C (left panel) and Myosin VIIa tail (right panel), the two proteins colocalize in cytoplasmic punctate structures (arrows). Bars, 10 µm. (G) 35S-labeled myosin VIIa fragments (1-4 in upper panel) were incubated with either GST-mShrm2MBR or GST alone (see blots in bottom panels). The 35S-labeled myosin VIIa tail binds to GST-mShrm2MBR but not to GST (two lanes on left side (1) of right blot). Fragments 2 and 4 (Myosin VIIa SH3/MyTH4/FERM and Myosin VIIa MyTH4/FERM, respectively) bind to GST-mShrm2MBR (lanes 2 and 4 in right blot), whereas the SH3-MyTH4 fragment does not (lane 3 in right blot). (H) Co-immunoprecipitation assay. HEK293 cells were co-transfected with plasmids encoding either the full-length myosin VIIa and Myc-tagged shroom2 (Myc-mShrm2) or the full-length myosin VIIa and Myc tag alone (Myosin VIIa) (middle panels). The same experiment was carried out using the untagged myosin VIIa tail (Myosin VIIa tail) instead of the entire protein (top panels). Upon immunoprecipitation with the anti-Myc antibody, immunoblots were probed with antibodies against myosin VIIa or shroom2 as indicated. Both the Myosin VIIa and Myosin VIIa tail co-immunoprecipitate with Myc-mShrm2 (bottom panel).





Right arrow Return to article