The Journal of Neuroscience, February 15, 2006, ():
Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells
J. Neurosci. Senften et al.
26: 2060
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Fig. S1. Characterization of PCDH15 antibodies and PCDH15 expression in hair cells. (a) Diagram of the PCDH15 protein showing the extracellular cadherin repeats (EC1-11), transmembrane domaine (TM), 4 proline rich motifs in the cytoplasmic domain and a PDZ-binding domain at the C-terminus. C1 and C2 indicate the protein domains that were used to generate antisera. (b) The antibody detected PCDH15 by western-blot in extracts from 293 cells transfected with a PCDH15 expression vector. No protein was detectable in untransfected cells. (c) The antibody detected PCDH15 in transfected cells by immunocytochemistry. PCDH15 (red) was detectable around the nucleus (blue) and at the cell surface (arrows). The staining around the nucleus presumably reveals PCDH15 in the endoplasmatic reticulum. (d) Cochlear whole mounts of P5 wild-type mice were stained with phalloidin to reveal F-actin (green) and with antibodies to PCDH15 (red). The image is a higher magnification view of some of the panels shown in Fig. 1. One row of inner hair cells (bottom) and two rows of outer hair cells (top) are shown. PCDH15 was expressed in stereocilia of inner and outer hair cells. Note that in outer hair cells, PCDH15 could frequently be visualized in two adjacent rows of stereocilia (arrows). Size bars: (c) 20 μm; (d) 5 μm.
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Supplementary Fig. S2. Cell polarity defects. (a,b) Cochlear whole mounts from P5 animals were stained with phalloidin to reveal hair bundles. The polarity of hair bundles was analyzed and is indicated by arrows pointing towards the longest stereocilia. Hair bundles in the Ames waltzerav3J mice (b) showed a slight polarity defect when compared to wild-type mice (a). (c) The polarity of hair cells was determined in the cochlear sensory epithelia from two wild-type and two Ames waltzerav3J mice. The axis running perpendicular to the long axis of the cochlear sensory epithelium was set at 0o. The data are expressed as mean ± SD. Size bar: 20 μm.
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Supplementary Fig. S3. PCDH15 binds to harmonin. (a) Diagram of the GFP-harmonin protein that were used for the biochemical experiments. (b) Cells were transfected with PCDH15 and the GFP-tagged harmonin construct indicated on top of the panel. Immunoprecipitations were carried out with GFP antibodies and extracts were resolved by SDS-PAGE. PCDH15 was visualized by western-blotting. Upper panel: PCDH15 co-immunopricipitated with harmonin-GFP, but not GFP alone. Middle and lower panel: Extracts were directly analyzed for the expression of PCDH15 and GFP-tagged harmonin without immunoprecipitation, to confirm that the transfected proteins were expressed to similar levels. (c) Cells were transfected with PCDH15 and the GFP-tagged harmonin constructs indicated on top of the panel. Immunoprecipitations were carried out with GFP antibodies and extracts were resolved by SDS-PAGE. PCDH15 was visualized by western-blotting. Upper panel: PCDH15 co-immunoprecipitated with constructs containing the PDZ-2 domain of harmonin. Middle and lower panel: Extracts were directly analyzed for the expression of PCDH15 and GFP-tagged harmonin constructs without immunoprecipitation, to confirm that the transfected proteins were expressed to similar levels. (c) The indicated GST-tagged PCDH15 constructs were used for GST pull-down experiments using in vitro translated harmonin. Upper panel: A protein containing part of the cytoplasmic domain of PCDH15 (GST-PCDH15CT) was able to interact with harmonin, while a similar protein lacking the C-terminal 8 amino acids (GST-PCDH15CTΔPBI) did not bind. GST alone also did not interact with PCDH15. As a control, the input PCDH15 prior to GST pull-down was loaded on the gel. Lower panel: Coomassie blue staining revealed that all GST construct were used in similar amounts.
