Supplemental Figure 1: Immunoelectron microscopy of ribeye on the harvested sucrose pellet from the gradient centrifugation shown in multiple panels. Supplemental Figure 2: Immunoelectron microscopy of VAMP2 on the harvested 200-400 mM sucrose interface sample shown in multiple panels. Supplemental Figure 3: A. Western blots indicate the immunoprecipitation of ribeye and CtBP2 with the CtBP2 antibody. Also coimmunoprecipitation of CtBP1, actin, myosin 9H, spectrin and α-tubulin is shown. B. Western blots indicate the immunoprecipitation of only ribeye with the AD antibody and the coimmunoprecipitation of PRPF39 is indicated. Supplemental Figure 4. Colocalization of ribeye with various presynaptic proteins. The labeling of individual proteins is presented in grayscale and merged panels are depicted in color. Double-labeling with ribeye-specific antiserum reveals the localization with respect to synaptic ribbons of SNARE proteins such as syntaxin 1 (A-C), SNAP25 (D-F), VAMP2 (G-I), syntaxin 6 (J-L), SNAP23 (M-O), VAMP7 (P-R), VAP-33 (S-U); SNARE dissociation molecules such as α-SNAP, β-SNAP (V-X), NSF (Y-A′); hair-cell specific proteins such as glutamate receptors 2 and 3 (GluR2 and GluR3) (B′-D′), glutamate transporter vGluT3 (E′-G′), hair-cell specific Ca2+ channel Cav1.3 (H′-J′), calcium sensor otoferlin (K′-M′); and other synaptic proteins such as rabphilin (N′-P′), munc 18 (Q′-S′), and synapsin (T′-V′). Supplemental Figure 5. Colocalization of ribeye with endocytotic and vesicle- trafficking proteins. The labeling of individual proteins is presented in grayscale and merged panels are depicted in color. Double-labeling with ribeye-specific antiserum reveals the localization of the endocytic proteins clathrin (A-C), dynamin (D-F), SCAMP1 (G-I) and endophilin (J-L) and of the trafficking proteins Sec13 (M-O), β-COP (P-R), VCP (S-U), and GAPDH (V-X) with respect to synaptic ribbons. Supplemental Figure 6. Colocalization of ribeye with cytoskeletal proteins. The labeling of individual proteins is presented in grayscale and merged panels are depicted in color. Double-labeling with ribeye-specific antiserum reveals the localization of the cytoskeletal proteins α-tubulin (A-C), spectrin (D-F), MYH9 (G-I), β-catenin (J-L), NF200 (M-O), and KIF3A (P-R) with respect to synaptic ribbons. Supplemental Table 1: Listing of antibodies and antisera used in immunoblotting and immunocytochemical experiments. Supplemental Tables 2-7: Identification of cochlear proteins from sucrose gradients. The isolated proteins are tabulated in alphabetical order. For each protein, the accession number and the protein score defining the total fragment ions matching score (MS/MS matches only) is shown. Protein scores are derived from ion scores as a non-probabilistic basis for ranking protein hits. The ion score is -10·log(P), in which P is the probability that the observed match is a random event. We used stringent criteria for protein identification: a protein was designated as a “hit” only if it matched at least two distinct peptides with an ion score of at least 40. A single-peptide match was considered if the peptide had a good tandem mass spectrum and matched only a single protein. Because we used the non-redundant NCBI (NCBInr) Chordata database to search the tandem mass-spectrometric data, the accession numbers represent different species; when data from multiple species are used, that with the highest score is used. Also shown are the protein's mass, the peptide matches that define the total number of peptides matched to that protein, the protein coverage giving the percentage of peptide sequences matched to that protein, and the isoelectric point (PI). Supplemental Table 2: Identification of proteins from the sucrose pellet in the cochlea. Supplemental Table 3: Identification of proteins from the sucrose pellet in the retina. Supplemental Table 4: Identification of proteins from the harvested sucrose gradients between the 200-400 mM interface from cochlear material. Supplemental Table 5: Identification of proteins from the harvested sucrose gradients between the 400-600 mM interface from cochlear material. Supplemental Table 6: Identification of proteins from the harvested sucrose gradients between the 600-800 mM interface from cochlear material. Supplemental Table 7: Identification of proteins from the 47,000 x g pellet fraction from cochlear material. EM negative staining of the sucrose pellet fraction with the AD antibody indicating ribeye staining A B C D Scale bar: 100 nm EM negative staining of the vesicle fraction between the 200-400 mM sucrose layers using VAMP2 antibody A B C D E F Scale bar: 100 nm A. Coimmunoprecipitation with CtBP2 antibody: experiment I and experiment II (results in Table 2) eads P l b kDa kDa Ab flowthPreolulegthSupernaFtelonwt througChontro CTBP2 I Ab flowPtehlrleotuSguhpernaFtleonwtthroEulguhtion1Elution2Elution3Beads 120 120 100 ribeye 100 ribeye 80 80 60 60 50 50 CtBP2 CtBP2 50 CtBP1 40 myosin 9H 220 120 50 50 40 actin spectrin 30 40 20 30 60 50 40 B. Coimmunoprecipitation with AD antibody (Table 3 results) l o l t h r o n nt tr o n co kDa h as Pellet SupernateFlowthroWug Elution1 Elution2 Beads Elution cBeads 120 Ribeye 100 80 60 50 IgG CtBP2 120 100 80 PRPF39 60 IgG 50 A ribeye B syntaxin 1 C Merge B’ ribeye C’ GluR 2/3 D’ Merge Phalloidin Phalloidin D ribeye E SNAP25 F Merge Phalloidin E’ ribeye F’ vGlut3 G’ Merge Phalloidin G ribeye H VAMP2 I Merge Phalloidin H’ ribeye I’ Cav1.3 J’ Merge J ribeye K syntaxin 6 L Merge Phalloidin Phalloidin K’ ribeye L’ Otoferlin M’ Merge M ribeye N SNAP23 O Merge Phalloidin Phalloidin P ribeye Q VAMP7 R Merge Phalloidin N’ ribeye O’ rabphilin P’ Merge Phalloidin S ribeye T VAP-33 U Merge Phalloidin V ribeye W X Merge Phalloidin Q’ ribeye R’ munc18 S’ Merge Phalloidin Y ribeye Z NSF A’ Merge Phalloidin T’ ribeye U’ synapsin V’ Merge Phalloidin A ribeye B clathrin C Merge M ribeye N Sec13 O Merge Phalloidin Phalloidin P ribeye Q R Merge Phalloidin D ribeye E dynamin F Merge Phalloidin S ribeye T VCP U Merge Phalloidin G ribeye H SCAMP1 I Merge Phalloidin J endophilin K ribeye L Merge V ribeye W GAPDH X Merge Phalloidin Phalloidin A B ribeye C Merge Phalloidin D spectrin E CtBP2 F Merge Phalloidin G myosin H9 H CtBP2 I Merge Phalloidin J ribeye K L Merge Phalloidin M NF200 N CtBP2 O Merge Phalloidin P KIF3A Q CtBP2 R Merge Phalloidin Supplemental Table 1. List of antibodies and antisera. Antibodies Company and catalog number Concentrations Immunoblot Immunocytochemistry Otoferlin (R), HCS1 J.T. Corwin’s Laboratory NA 1:500 bassoon (M) Abcam ab76065 1:1000 1:500 NIPSNAP1 (R) Abcam ab67302 NA 1:200 SNAP25 (R) Abcam ab24737 1:1000 NA SNAP25 (m) Synaptic Systems 111 011 NA 1:200 VAMP1 (R) Abcam ab3346 1:2000 1:250 VAMP7 (M) Abcam ab36195 NA 1:100 clathrin heavy chain (M) Affinity Bioreagents MA1-065 1:500 1:250 VAMP2 (R) Assay Designs VAS-SV006F 1:2000 NA VAMP2 (M) Stressgen VAMSV014E NA 1:250 CASK (M) BD Transduction Laboratories 610782 1:500 NA CtBP1 (M) BD Transduction Laboratories 612042 1:2000 1:500 CtBP2 (M) BD Transduction Laboratories 612044 1:1000 1:500 dynamin (M) BD Transduction Laboratories 610246 1:1000 1:500 munc13 (M) BD Transduction Laboratories 610998 1:1000 NA munc18 (M) BD Transduction Laboratories 610337 1:1000 1:100 N-cadherin BD Transduction Laboratories 610920 1:2500 NA Rab3 (M) BD Transduction Laboratories 610380 1:2500 1:250 Rabphilin (M) BD Transduction Laboratories 610509 NA 1:250 RIM1 (M) BD Transduction Laboratories 610906 1:500 NA SCAMP1 (M) BD Transduction Laboratories 612087 NA 1:100 synapsin 1 (R) BD Transduction Laboratories 611392 1:10,000 NA Synapsin 1 (R) Hudspeth Laboratory NA 1:200 synaptotagmin1 (M) BD Transduction Laboratories 610434 1:500 NA syntaxin 4 (M) BD Transduction Laboratories 610439 1:10,000 NA syntaxin 6 (M) BD Transduction Laboratories 610635 1:2500 1:200 tomosyn (M) BD Transduction Laboratories 611297 1:500 NA VAP-33 (M) BD Transduction Laboratories 612180 1:2000 1:100 VCP97 (M) BD Transduction Laboratories 612182 NA 1:250 β-Catenin (M) BD Transduction Laboratories 610154 1:500 1:500 CaMKII (R) Cell Signalling 3361S NA 1:50 Complexin 1/2 (R) Chemicon International AB9288 1:1000 NA gluR 2 and 3 (R) Chemicon International AB 1506 NA 1:100 SV2 (M) Developmental Hybridoma Bank 1:1000 NA GAPDH (M) Millipore MAB374 1:1000 1:1000 GFAP (M) Novus Biologicals NB 300-141 1:1000 NA Sec13 (M) Novus Biologicals H00006396-A01 NA 1:100 β-COP (R) Pierce Biotechnology PA1-061 NA 1:100 CaBP4 (G) Santa Cruz Biotechnology sc-28096 1:500 1:500 synaptophysin 1 (R) Santa Cruz Biotechnology sc-9116 1:500 1:500 α-SNAP, β-SNAP (M) Santa Cruz Biotechnology sc-48349 1:250 1:100 actin (R) Sigma A 2066 1:500 1:250 KIF3A (R) Sigma K 3513 1:1000 1:200 Myosin H9 (R) Sigma M 8064 NA 1:500 NF160 (M) Sigma N 5264 1:1000 NA NF200 (R) Sigma N 4142 1:1000 1:250 NF68 (M) Sigma N 5139 1:1000 NA NSF (M) Sigma N 6038 1:2000 1:500 SNAP23 (R) Sigma Prestige Andtibodies HPA001214 1:2000 1:200 syntaxin 1 (M) Sigma S 0664 1:2000 1: 250 Synaptophysin (M) Sigma S 5768 Syntaxin 7 (R) Sigma S 4819 1:1000 NA α-tubulin (M) Sigma T 6074 1:2000 1:500 γ-tubulin (M) Sigma T 3559 1:10,000 NA Spectrin (M) Sigma Immunochemicals S1390 NA 1:200 PRPF39 (R) Sigma Prestige Antibodies HPA001176 NA 1:100 CSP (R) Stressgen VAP-SV003 1:5000 NA ERC2 (R) Synaptic Systems 143 103 1:1000 NA Piccolo (R) Synaptic Systems 142 002 1:2000 1:200 RIM2 (R) Synaptic Systems 140 103 1:1000 NA synaptojanin (R) Synaptic Systems 145 003 1:1000 NA VAMP3 (R) Synaptic Systems 104 103 1:2000 1:200 Cav1.3 (R) Teresa