Suzanne de Bruijn

122 Chapter 3.1 Injectoporation of Ripor2 -constructs, immunostaining and antibodies For injectoporation, the organ of Corti was isolated and placed in DMEM/F12 medium with 1.5 µg/ml ampicillin. Glass electrodes (~2 µm diameter) were used to deliver the plasmid (500 ng/µl in Hank's Balanced Salt Solution (HBSS)) to the sensory epithelium. A series of 3 pulses were applied at 1 sec intervals with a magnitude of 60V and duration of 15 msec (ECM 830 square wave electroporator; BTX). Two days after injectoporation, samples were fixed in the fixative containing 4% paraformaldehyde in HBSS for 20 min. Tissues were then washed in HBSS and blocked for 20 min at room temperature in HBSS containing 5%BSA, 1%goat serumand 0.5%Triton X-100, and then incubated overnight at 4 o Cwith primary antibodies in HBSS containing 1%BSA and 0.1%Triton X-100. Tissues were washed in HBSS and incubated 2 hours at room temperature with secondary antibodies. Tissues were mounted in ProLong ® Antifade Reagents (ThermoFisher). Stacked images were then captured by fluorescence deconvolution microscope (Leica). Antibodies used were: anti-HA (mouse; 1:500; cat.#2367S; Cell Signaling), Alex Fluor 568-phalloidin (1:500; cat.#A12380; ThermoFisher) and Alexa Fluor 488 goat anti-mouse (1:1000; cat.#A11017; ThermoFisher). Antibodies used for co-immunoprecipitations were: anti-HA (mouse; 1:500; cat.#2367S; Cell Signaling), anti-Myc (rabbit; 1:500; cat.#2278S; Cell Signaling), anti-Myc (mouse; 1:500; cat.#9E10; Santa Cruz); anti-GFP (mouse; 1:1000; cat.#SC-9996; Santa Cruz). SUPPLEMENTARY RESULTS The RIPOR2 c.1696_1707del variant is derived from a common ancestor VNTR marker analysis was performed to determine whether a haplotype of the chromosomal region flanking the variant was shared by the different families. Indeed, a shared haplotype of ~1.0 Mb, delimited by markers D6S2439 and D6S1281, was found in the seven families for which segregation analysis of the marker alleles could be performed ( Figure S3 ). This haplotype was also potentially shared by the five single cases. For marker D6S1545, a different CA-repeat length was determined on the variant- carrying allele of familyW18-0470 whereas the alleles of two more centromeric markers where still shared. Since a rare event that caused a repeat length change of the D6S1545 allele may have occurred, this marker locus was still considered to be part of the shared haplotype. To further refine the shared haplotype, we extracted homozygous SNPs present in the region between D6S2439 and D6S1281 from the exome sequencing datasets. Subsequently, homozygous SNP genotypes were compared between all