(A) Immunostaining of WT cochlea or PCDH15 KO with polyclonal antibody raised in rabbits against PCDH15 extracellular domain. Scale bars correspond to 10 µm. (B) Immunostaining of WT utricle with …
(A) FSEC analysis of binding of 39G7 to the PCDH15 extracellular domain. 39G7 addition leads to earlier elution of PCDH15 in full-length constructs and after deletion of EC1 and EC2. Deletion of EC3 …
Original image of polyacrylamide gel shown in Figure 2C.
(A–D) Representative examples of PCDH15 dimers with two bound AuNPs (A, B) or one bound AuNP (C, D) image in stereocilia. On the left-hand side of each panel, a manually annotate model is shown in a …
(A) Representative example of stereocilia tip with PCDH15 molecule only found in adjacent shaft. (B, C) Representative examples of stereocilia tips with one copy of PCDH15 at tip. (D, E) …
On the left side of each panel, the tomogram annotation is shown from the side and from top. On the sideview ice thickness is indicated. To the right of the annotation is a projection of the cluster …
(A) Annotation of tomogram showing a putative intact tip link. The inset at the top left shows a projection of the tomogram. (B) Close-up view of putative tip link density in the tomogram. (C) …
The tip also contains PCDH15 dimers not bound to a complex partner. An additional PCDH15-complex is connecting the two stereocilia in the shaft region. Also depicted in Figure 6.
(A) Annotation of tomogram showing developing stereocilia including PCDH15-containing ‘lateral links.’ The inset at the bottom right shows a projection of the tomogram. (B, C) Close-up of putative …
Noteworthy complexes that include apparent interaction between putative CDH23 dimers, as well as bends in the putative CDH23 dimer are highlighted. Also depicted in Figure 7.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Mus musculus) | Pcdh15 | Uniprot | Q99PJ1 | |
Cell line (Spodoptera frugiperda) | Sf9 | Thermo Fisher Scientific | 12659017 RRID:CVCL_0549 | |
Cell line (Homo sapiens) | HEK293 tsa 201 | ATCC | CRL-11268RRID:CVCL_1926 | |
Recombinant DNA reagent | PCDH15 EC1-EL-YFP expression plasmid | This paper | Created using pEG BacMam (Addgene plasmid # 160451) | |
Recombinant DNA reagent | 39G7 Fab’ expression plasmid | This paper (created by VectorBuilder) | Backbone described in d oi:10/fhwrn3 | |
Antibody | Anti-PCDH15 (Rabbit polyclonal) | This paper (created by Genscript) | IF: 10 µg/ml | |
Antibody | Anti-PCDH15 39G7 (Rabbit monoclonal) | This paper (created by Genscript) | IF: 10 µg/ml | |
Biological sample (M. musculus) | Inner ear | P6–P9 mice | ||
Chemical compound, drug | 10 nm gold fiducials | Ted Pella | 90010 | |
Chemical compound, drug | HAuCL4 | Sigma-Aldrich | 520918 | |
Chemical compound, drug | 3-MBA | Sigma-Aldrich | 451436 | |
Chemical compound, drug | mPEG-550-SH | Creative PEGworks | PLS-607 | |
Software, algorithm | SerialEM | 10.1016 /j.jsb.2005.07.007 | RRID:SCR_017293 | |
Software, algorithm | IMOD | doi.org.10.1006/jsbi.1996.0013 | RRID:SCR_003297 | |
Software, algorithm | Topaz | doi.org.10.1038/s41467-020-18952-1 | ||
Software, algorithm | TomoAlign | doi.org.10.1016/j.jsb.2019.01.005 | ||
Software, algorithm | Motioncor2 | doi.org.10.1038/nmeth.4193 | RRID:SCR_016499 | |
Software, algorithm | ChimeraX | doi.org.10.1002/pro.3943 | RRID:SCR_015872 |