Suzanne de Bruijn

143 A RIPOR2 deletion is a frequent cause of adult-onset hearing loss RIPOR2 wildtype allele <20 years 20-40 years 40 years ≥ Controls 0 20 40 60 80 Relative expression to GUSB RIPOR2 mutant allele <20 years 20-40 years 40 years ≥ Controls 0 10 20 30 40 RIPOR2 total expression <20 years 20-40 years 40 years ≥ Controls 0 10 20 30 40 50 Ratio RIPOR2 mutant/wildtype <20 years 20-40 years 40 years ≥ Controls 0.0 0.5 1.0 1.5 2.0 A B * Relative expression to GUSB Relative expression to GUSB Ratio mutant/wildtype RIPOR2 Figure S7. Transcript levels of RIPOR2 alleles determined by RT-qPCR. (A) Subjects were divided in three groups based on the reported ages of onset: early onset (<20 years, n=7), middle onset, (20-39 years, n=15) and late onset (≥40, n=6) hearing impairment. RNA samples isolated from peripheral blood of individuals without the RIPOR2 variant were used as controls (n=10). (B) Calculated ratio of RIPOR2 mutant to wildtype relative expression analysis. A one-way ANOVA followed by Tukey’s multiple comparison test was employed to identify potentially significant differences between the transcript levels of the groups. * p-value = 0.0214. A Myc-RIPOR2wt HA-RIPOR2wt HA-RIPOR2mut Co-IP: Myc Co-IP: HA Input: Myc _ + + + _ + + _ _ B RIPOR2wt-GFP RIPOR2mut-GFP HA-RHOC Co-IP: GFP Co-IP: HA Input: GFP _ + + _ + + _ + _ _ + _ Input: HA Input: HA Figure S8. RIPOR2 dimerization and interaction with RHOC. (A) Interaction of murine RIPOR2-wildtype (RIPOR2wt) and -mutant (RIPOR2mut) was studied using CoIP assays. HEK293T cells were transfected with constructs encoding N-terminally tagged proteins as indicated above each panel. Immunoprecipitations were performed using anti-HA antibodies, followed by western blotting. (B) Interaction with RHOC was studied using C-terminally GFP-tagged RIPOR2-wildtype or -mutant and N-terminally HA-tagged RHOC. Immunoprecipitations were performed using anti-HA antibodies, followed by western blotting.