Skip to main content
. 2016 Aug 15;5:e18312. doi: 10.7554/eLife.18312

Figure 3. MYO7A immunoaffinity purification from D10 enriched membranes.

(A) Flow chart describing immunoaffinity purification strategy using the 138-1 monoclonal antibody, which recognizes chicken MYO7A. Mass spectrometry was carried out on the total (S7; RIPA-soluble fraction), 138-1 eluate, and control eluate. F/T, unbound material; MS/MS, tandem mass spectrometry analysis. (B–E) Shotgun proteomics analysis of 138-1 immunoaffinity purification. (B) Relative protein levels of 1719 proteins detected in at least one run of total or 138-1 eluate. Distance from the diagonal unity line (gray dashed) indicates enrichment by 138-1; note that MYO7A (red symbol) and PDZD7 (green) are highly enriched. (C) Volcano plot illustrating enrichment and statistical significance. The x-axis displays the log10 of each protein’s enrichment by 138-1 immunoprecipitation relative to the S7 starting material, while the y-axis indicates the log10 value of the FDR (false discovery rate) adjusted p-value for that enrichment value. Dashed line indicates significance at p=0.05. (D) Top 32 proteins in 138-1 eluates by enrichment. Only proteins not detected in control runs and detected in at least four of six 138-1 runs are displayed. (E) Top 27 proteins in 138-1 eluates by stoichiometry relative to MYO7A. Only proteins detected in at least four of six 138-1 runs are displayed; proteins detected in control eluates (e.g., actin) were also included. Unadjusted stoichiometry: (138-1 riBAQ for protein of interest)/(138-1 riBAQ for MYO7A). Adjusted stoichiometry: (unadjusted stoichiometry) × (total riBAQ for MYO7A)/(total riBAQ for protein of interest). Figure 3—figure supplement 1 displays an immunoblot analysis of the 138-1 immunoprecipitation, examining MYO7A and its Usher syndrome partners USH1C and CDH23.

DOI: http://dx.doi.org/10.7554/eLife.18312.011

Figure 3—source data 1. Analysis of the shotgun proteomics experiments characterizing the protein composition of the 138-1 anti-MYO7A immunoaffinity purification from the chick inner ear.
DOI: http://dx.doi.org/10.7554/eLife.18312.012
elife-18312-fig3-data1.xls (2.3MB, xls)
DOI: 10.7554/eLife.18312.012

Figure 3.

Figure 3—figure supplement 1. Immunoprecipitation of upper tip-link density components from D10-enriched stereocilia membranes.

Figure 3—figure supplement 1.

(A) MYO7A (asterisk). IP input was 100%. Key: IP input, ears starting total extract (S7); C, control immunoprecipitates; 7A, 138-1 immunoprecipitates; IPs, immunoprecipitates. (B) USH1C (asterisk). IP input was 100%. (C) CDH23. IP input was 4 ear-equivalents, which corresponds to 100% (4 ears) and 20% (19 ears). With image enhancements to the blot, CDH23 bands were apparent in 138-1 eluates but not in control eluates. Key: Pre-D10, dense membranes (P2); ears, ear-equivalents loaded; Enhanced, blot adjusted by changes to dark level and gamma. Asterisks indicate positions of high (*) and lower (**) mass bands immunoreactive for CDH23.
DOI: http://dx.doi.org/10.7554/eLife.18312.013